Compositions and methods related to human neutralizing antibodies to hepatitis b

ABSTRACT

Provided are broadly neutralizing antibodies (bNAbs) and antigen binding fragments thereof that bind with specificity to epitopes expressed by Hepatitis B vims (HBV). The bNAbs target non-overlapping epitopes on the HBV S antigen (HBsAg). Pharmaceutical compositions that contain the bNAbs, or modified bNAbs, are provided. Combinations of the bNAbs are included, and are useful for prophylaxis and therapy of HBV infection, and for inhibiting development of HBV escape mutations in infected individuals. Expression vectors encoding the bNAbs and antigenic fragments of them are included, as are methods of making the bNAbs and antigenic fragments of them. HBV peptides for use as vaccines are provided, and include at least two non-overlapping epitopes from the HBsAg. Diagnostic reagents comprising the bNAbs or antigenic fragments thereof are provided, as are methods of detecting HBV and diagnosing HBV infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent application No. 62/898,735, filed Sep. 11, 2019, and to U.S. provisional patent application no. 62/982,276, filed Feb. 27, 2020, the entire disclosures of each of which are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant no. UL1TR001866 awarded by The National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 9, 2020, is named 076091_00092_SL.txt and is 307,317 bytes in size.

BACKGROUND

Despite the existence of effective vaccines, hepatitis B virus (HBV) infection remains a major global health problem with an estimated 257 million people living with the infection. Whereas 95% of adults and 50-75% of children between the ages of 1 and 5 years spontaneously control HBV, only 10% of infants recover naturally. The remainder develop a chronic infection that can lead to liver cirrhosis and hepatocellular carcinoma. Although chronic infection can be suppressed with antiviral medications, there is no effective curative therapy (Dienstag, 2008; Revill et al., 2016; Thomas, 2019).

HBV is an enveloped double-stranded DNA virus of the Hepadnaviridae family. Its genome is the smallest genome among pathogenic human DNA viruses, with only four open reading frames. Infected liver cells produce both infectious HBV virions (Dane particles) and non-infectious subviral particles (Australia antigen) (Dane et al., 1970; Hu and Liu, 2017). The virion is a 42 nm-diameter particle containing the viral genome and HBV core antigen (HBcAg) encapsidated by a lipid membrane containing the hepatitis B surface antigen (HBsAg) (Blumberg, 1964; Venkatakrishnan and Zlotnick, 2016). Subviral particles lack the viral genome.

HBV strains were originally grouped into four HBsAg serotypes (adr, adw, ayw, and ayr). Genetic analysis revealed several highly conserved domains and defined eight genotypes A-H, which are highly correlated with the 4 serotypes (Norder et al., 2004). The HBV surface protein, HBsAg, has 4 putative transmembrane domains and can be subdivided into PreS1-, PreS2- and S-regions. The S domain is a cysteine-rich protein consisting of 226 amino acids that contain all 4 of the transmembrane domains (Abou-Jaoude and Sureau, 2007). In addition, the S-protein can be glycosylated at asparagine residue 146 (Julithe et al., 2014).

Antibodies to HBsAg (anti-HBs) are associated with successful vaccination and recovery from acute infection, while antibodies to HBcAg (anti-HBc) are indicative of past or current HBV infection (Ganem, 1982). Indeed, the most significant difference between chronically infected and naturally recovered individuals is a robust antibody response to HBsAg (Ganem, 1982). Conversely, the inability to produce these antibodies during acute infection is associated with chronicity (Trepo et al., 2014). Whether these associations reflect an etiologic role for anti-HBs antibodies in protecting from or clearing established infection is not known. However, depletion of antibody-producing B lymphocytes in exposed humans by anti-CD20 therapies (e.g. rituximab) is associated with HBV reactivation, indicating that B cells and/or their antibody products play a significant role in controlling the infection (Loomba and Liang, 2017).

Several human antibodies against HBsAg have been obtained using a variety of methods including: phage display (Kim and Park, 2002; Li et al., 2017; Sankhyan et al., 2016; Wang et al., 2016); humanized mice (Eren et al., 1998); Epstein-Barr virus-induced B cell transformation (Heijtink et al., 2002; Heijtink et al., 1995; Sa'adu et al., 1992); hybridoma technology (Colucci et al., 1986); human B cell cultures (Cerino et al., 2015); and microwell array chips (Jin et al., 2009; Tajiri et al., 2010). However, the donors in these studies were not selected for serum neutralizing activity. Thus, there remains a need for improved approaches and compositions of combatting HBV infection. The present disclosure is pertinent to this need.

BRIEF SUMMARY

The disclosure provides in part a description of the human humoral immune response to HBsAg in immunized and spontaneously recovered individuals that had been selected for high levels of serum neutralizing activity. The disclosure demonstrates that these individuals develop closely related bNAbs that target shared non-overlapping epitopes in HBsAg. The crystal structure of one of the antibodies with its peptide target reveals a loop that helps to explain why certain amino acid residues are frequently mutated in escape viruses and why combinations of bNAbs may be needed to control infection. In vivo experiments in humanized mice demonstrate that the bNAbs are protective and can be therapeutic when used in combination.

Any antibody described herein can comprise at least one modification of its constant region. The modification may be made for any one or more amino acids. The modification can have any of a number of desirable effects. In certain approaches, the modification increases in vivo half-life of the antibody, or alters the ability of the antibody to bind to Fc receptors, or alters the ability of the antibody to cross placenta or to cross a blood-brain barrier or to cross a blood-testes barrier, or inhibits aggregation of the antibodies, or a combination of said modifications, or wherein the antibody is attached to a label or a substrate. In embodiments, the modification improves the manufacturability of the antibody. In embodiments, any antibody or combination thereof described herein can be present in an immunological assay, such as an enzyme-linked immunosorbent assay (ELISA) assay, or an ELISA assay control. The ELISA assay can be any of a direct ELISA assay, an indirect ELISA assay, a sandwich ELISA assay, or a competition ELISA assay.

In another aspect the disclosure provides a method for prophylaxis or therapy of a hepatitis viral infection comprising administering to an individual in need thereof an effective amount of at least one antibody described herein, or an antigen binding fragment thereof. The antibody may comprise at least one modification of the constant region. In embodiments, the composition is administered to an individual who is infected with or is at risk of being infected with a hepatitis B virus. In one approach, at least two antibodies are administered, wherein optionally the two antibodies recognize distinct HBV epitopes. In an embodiment, administering at least two distinct antibodies suppresses formation of viruses that are resistant to the antibodies.

In another aspect the disclosure provides vaccine formulations. In an embodiment a vaccine formulation comprises an isolated or recombinant peptide or a polynucleotide encoding the peptide, wherein the peptide is derived from an epitope that is frequently targeted by HepB immune resistance, and which is located in a loop anchored by oppositely charged residues, as further described herein.

In another aspect the disclosure provides one or more recombinant expression vectors, and kits comprising the expression vectors. The expression vectors encode at least the heavy chain and the light chain CDRs of any of the antibodies of described herein. Cells comprising the recombinant expression vectors are included, as are methods of making antibodies by culturing cells that comprise expression vectors that express the antibodies, and separating antibodies from the cells. Cell culture media containing such cells and/or antibodies is also included.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Antibody responses in HBV vaccinated and recovered individuals. (A) Donor screen. Sera from 159 volunteers were evaluated for anti-HBs binding by ELISA (x-axis) and HBV serum neutralization capacity using HepG2-NTCP cells (y-axis). Serum neutralization capacity on the y-axis was calculated as the reciprocal of the relative percentage of infected HepG2-NTCP cells. The values for unexposed naïve donors are Neutralization tests were performed at 1:5 serum dilution in the final assay volume. Each dot represents an individual donor. Green indicates unvaccinated and unexposed, black indicates vaccinated, and red indicates spontaneously recovered. The dashed line indicates the no serum control. Top neutralizers (serum neutralization capacity higher than 4) are indicated (top right). Boxed are representative samples shown in FIG. 2A. Spearman's rank correlation coefficient (r_(s)) and significance value (p). (B and C) Dose-dependent HBV neutralization by serum (B) or by purified IgG (C). Two assays were used to measure percent infection: ELISA to measure HBsAg protein in the medium (upper panels) and immunofluorescent staining for HBcAg in HepG2-NTCP cells (lower panels). Dashed line indicates virus-only control. (D) Schematic representation illustrating the three forms of the HBV surface protein: L-, M- and S-protein. These three forms of envelope protein all share the same S-region, with PreS1/PreS2 and PreS2 alone as the N-terminal extensions for L- and M-protein, respectively. (E)S-protein produced in Chinese hamster ovary (CHO) cells blocks serum neutralizing activity. Graphs show infection efficiency as a function of the amount of S-protein added. The concentration of polyclonal IgG antibodies (pAb) is indicated. Upper and lower panels are as in (B) and (C). A representative of at least two experiments is shown. See also FIG. 8 and Table S1.

FIG. 2. S-protein-specific antibodies. (A) Frequency of S-protein-specific memory B cells. Representative flow cytometry plots displaying the percentage of all IgG⁺ memory B cells that bind to both allophycocyanin- and phycoerythrin-tagged S-protein (S-protein-APC and S-protein-PE). Flow cytometry plots from other individuals are shown in FIG. 9A. Experiments were repeated two times. (B) Dot plot showing the correlation between the frequency of S-protein-binding IgG⁺ memory B cells and the serum neutralizing activity. Spearman's rank correlation coefficient (r_(s)) and significance value (p). (C) Each pie chart represents the antibodies from an individual donor, and the total number of sequenced antibodies with paired heavy and light chains is indicated in the center. Antibodies with the same combination of IGH and IGL variable gene sequences and closely related CDR3s in each individual are shown. The slices with the same color indicate shared antibodies with the same or similar combination of IGH and IGL variable genes between individuals (FIG. 9B). Grey slices indicate antibodies with closely related sequences that are unique to a single donor. In white are singlets. (D) V(D)J alignments for representative IGHV3-30/IGLV3-21, IGHV3-33/IGLV3-21 and IGHV3-23/IGLV3-21 antibodies from donors #60/#146 (H006 and H008), #146/#13 (H014 and H012), and #13/#60/#146 (H021, H003 and H004) respectively. Boxed grey residues are shared between antibodies. See also FIG. 9 and Table S2. Figure discloses SEQ ID NOS 1438-1451, respectively, in order of appearance.

FIG. 3. Broad cross-reactivity. (A) Binding to S-protein (adr serotype). 50% effective concentration (EC₅₀ in ng/ml) required for binding of the indicated human monoclonal antibodies to the S-protein. Libivirumab (Eren et al., 2000; Eren et al., 1998) and anti-HIV antibody 10-1074 (Mouquet et al., 2012) were used as positive and negative controls, respectively. All antibodies were tested. (B) Comparative binding of the mature and unmutated common ancestor (UCA) of antibodies H006, H019, and H020 to S-protein by ELISA. (C) Anti-HBs antibody binding to 5 different serotypes of HBsAg. Similar to panel (A), EC₅₀ values are color-coded: red, ≤50 ng/ml; orange, 50 to 100 ng/ml; yellow, 100 to 200 ng/ml; and white, >200 ng/ml. The abbreviation b.d. indicates below detection. All antibodies were tested. All experiments were performed at least two times. See also FIG. 10.

FIG. 4. HBsAg epitopes. (A) Competition ELISA defines 3 groups of antibodies. Results of competition ELISA shown as percent of binding by biotinylated antibodies and illustrated by colors: black, 0-25%; dark grey, 26-50%; light grey, 51-75%; white, >76%. Weak binders (H002, H012, H013, H014, H018) were excluded. Representative of two experiments. (B) Results of ELISA on alanine scanning mutants of S-protein. Only the amino acids vital for antibody binding are shown. Binding to mutants relative to wild-type S-protein: black, 0-25%; dark grey, 26-50%; light grey, 51-75%; white, >75%. Additional details are provided in FIG. 11. (C) Results of ELISA on human escape mutations of S-protein. Wild-type S-protein and empty vector serve as a positive and negative controls, respectively. Binding to mutants relative to wild-type S-protein: black, 0-25%; dark grey, 26-50%; light grey, 51-75%; white, >75%. Amino acid mutations in bold represent frequently observed mutations in humans (Ma and Wang, 2012). The antibodies tested in (B and C) were selected from Group-I, -II, -III based on their neutralizing activity (FIG. 5A-5C). All experiments were performed at least two times. See also FIG. 11.

FIG. 5. In vitro neutralization by the monoclonal antibodies. (A and B) In vitro neutralization assays using HepG2-NTCP cells. Percent infection in the presence of the indicated concentrations of bNAbs measured by ELISA of HBsAg in medium (A) and anti-HBcAg immunofluorescence (B). Anti-HIV antibody 10-1074 (Mouquet et al., 2012) and libivirumab (Eren et al., 2000; Eren et al., 1998) were used as negative and positive controls respectively. The corresponding IC_(50s) are shown in the left and middle column of panel (C). All experiments were repeated a minimum of two times. (C) bNAb 50% maximal inhibitory concentration (IC₅₀) calculated based on HBsAg ELISA (left column) and HBcAg immunofluorescence (middle column) for the in vitro neutralization assays using HepG2-NTCP cells, or HBeAg ELISA (right column) for in vitro neutralization using primary human hepatocytes. The abbreviation b.d. and n.d. indicate below detection and not done respectively. (D) In vitro neutralization using primary human hepatocytes. The levels of HBeAg in medium were measured by ELISA. The calculated IC₅₀ values are shown in the right column of panel (C). Experiments were repeated three times. (E) In vitro neutralization assay using HepG2-NTCP cells. IgG antibodies were compared to their corresponding Fab fragments. Concentrations of Fab fragments were adjusted to correspond to IgG. Experiment was performed two times. See also FIG. 12.

FIG. 6. Crystal structure of H015 bound to its recognition motif. A single crystal was used to obtain a high resolution (1.78 Å) structure. (A) Synthetic peptides (SEQ ID NOS 1452-1455, respectively, in order of appearance) spanning the antigenic loop region were subjected to ELISA for antibody binding. Among the tested antibodies, only H015 binds peptides-11 and -12. Experiments were performed three times and details are in FIG. 13A. (B and C) The peptide binds to CDR1 (R31), CDR2 (W52 and F53) and CDR3 (E99, P101, L103, and L104) of H015 heavy chain (green) and CDR3 (P95) of the light chain (cyan) (B). The interacting residues (C) on the heavy chain (green) are R31 (main chain), W52, F53 (main chain), E99, P101 (main chain), L103 (main chain), L104 (hydrophobic). One contact with the light chain (cyan) is with P95. (D) Electron density map of the bound peptide as seen in the 2Fo-Fc map contoured at 1 RMSD indicating high occupancy (92%). (E) The recognition motif, KPSDGN (SEQ ID NO: 1), adopts a sharp hairpin conformation due to the salt-bridge between K141 and D144 and is facilitated by kinks at P142 and G145. Glycine 145 (G145, circled) is the residue that escapes the immune system when mutated to an arginine. See also FIG. 13.

FIG. 7. Anti-HBs bNAbs are protective and therapeutic in vivo. (A and E) Diagram of the prophylaxis and treatment protocols, respectively. (B) Prophylaxis with isotype control antibody 10-1074 (Mouquet et al., 2012). (C and D) Prophylaxis with H020 and H007 respectively. The dashed line in (B-D) indicates the detection limit. (F) Treatment of viremic huFNRG mice with control antibody 10-1074. (G and H) Treatment of viremic huFNRG mice with H020 alone or H007 alone, respectively. HBV DNA levels in serum were monitored on a weekly basis. Two independent experiments comprising a total of 5 to 8 mice were combined and displayed. (I) Mutations in the S-protein sequence from the indicated mice (red arrows) in (G), (H) and (J). S-protein sequence chromotograms are shown in FIG. 14. (J-L) Treatment of viremic huFNRG mice with combination of anti-HBs bNAb H006+H007 (J), or H017+H019 (K), or H016+H017+H019 (L), respectively. Sequencing showed that none of the mice in (K) and (J) carried viruses with escape mutations in the S-protein. See also FIG. 14.

FIG. 8. Characterization of Antibody Immune Response Against HBV, Related to FIG. 1. (A) Schematic representation of different stages of HBV infection. Vaccinated or infected naturally recovered individuals were recruited for this study. (B) Sera (1:50 dilution in the final assay volume) from 159 volunteers were screened, see also FIG. 1A. (C-E) Comparison of anti-HBs ELISA titers (upper panel) and their serum neutralization capacity (lower panel) between different groups of individuals. Vaccinated or recovered individuals show statistically higher anti-HBs titers (upper panel, C) and more potent neutralizing activity (lower panel, C) than the uninfected unvaccinated individuals. Younger individuals (≤45 years old) showed slightly higher antibody immune response against HBsAg (D). No difference was found between genders (E).

FIG. 9. Antibody Cloning and Sequence Analysis of Anti-HBs, Related to FIG. 2. (A) Frequency of S-protein-specific memory B cells in peripheral blood mononuclear cells of all twelve donors. Details are similar to FIG. 2A. (B) Pie charts show the distribution of anti-HBs antibodies. Figure legends are similar to FIG. 2C. VH and VL genes for each slice are shown and the 20 chosen anti-HBs antibodies are labeled. (C) Phylogenetic tree of all cloned anti-HBs antibodies based on IGH Fab region. IGH Fab regions from 244 memory B cells sorted with HBsAg were aligned followed by tree construction.

FIG. 10. Autoreactivity of 20 anti-HBs antibodies, Related to FIG. 3. (A) Autoreactivity of monoclonal antibodies. Positive control antibody efficiently stained the nucleus of HEp-2 cells. Twenty anti-HBs antibodies, as well as anti-HBs antibody libivirumab and anti-HIV antibody 10-1074, were also tested. (B) Polyreactivity profiles of 20 anti-HBs antibodies. ELISA measures antibody binding to the following antigens: double-stranded DNA (dsDNA), insulin, keyhole limpet hemocyanin (KLH), lipopolysaccharides (LPS), and single-stranded DNA (ssDNA). Red and green lines represent positive control antibody ED38 and negative control antibody mGO53 respectively, while dashed lines show cut-off values for positive reactivity (Gitlin et al., 2016).

FIG. 11. Alanine Scanning and Peptide Screening, Related to FIG. 4. (A) Results of ELISA on alanine scanning mutants of HBsAg. Binding to mutants was normalized to wild-type S-protein: black, 0-25%; dark grey, 26-50%; light grey, 51-75%; white, >76%. Experiments were performed three times. Underlined cysteines, alanines, and amino acids known to be critical for S-protein production were not mutated (Salisse and Sureau, 2009). Figure discloses SEQ ID NO: 1456. (B) Schematic diagram of alanine scanning results. Figure discloses the primary amino acid sequence as SEQ ID NO: 1456 and the sequence containing alanine mutations as SEQ ID NO: 1457.

FIG. 12. In Vitro Neutralization Assay of anti-HBs bNAb Unmutated Common Ancestor Antibodies or Combinations, Related to FIG. 5. (A-B) In vitro neutralization assay of anti-HBs bNAbs and their corresponding unmutated common ancestor (UCA) antibodies. The relative infection rates were calculated based on either HBsAg protein level in culture medium (A) or HBcAg staining intracellularly (B). (C) In vitro neutralization assay of anti-HBs bNAbs recognizing different epitopes and the same total amount of antibody combination at 1:1 or 1:1:1 ratio.

FIG. 13. Detailed Information of Crystal Structure of H015 and Its Linear Epitope, Related to FIG. 6. (A) Synthesized peptides (SEQ ID NOS 1458-1476, respectively, in order of appearance) for antigenic loop region were subjected to ELISA for antibody binding. Among the tested antibodies, only H015 binds peptide-11 and -12. (B) Data collection and refinement statistics for H015 Fab are summarized. Statistics for the highest-resolution shell are shown in parentheses. Refinement program PHENIX 1.16. (C) The green/red density is the unbiased omit map. Red is negative density equated to noise. (D) Table of contacts within the peptide and between Fab fragment and peptide.

FIG. 14. HBV DNA levels and S-protein Sequences in Antibody-Treated huFNRG Mice, Related to FIG. 7. (A) HBV DNA levels in representative individual huFNRG mice treated by control antibody 10-1074, anti-HBs bNAb H020, anti-HBs bNAb H007, combination of anti-HBs bNAb (H006+H007), (H017+H019), and (H016+H017+H019). HBV DNA levels in mouse sera were monitored on a weekly basis. The mice without arrows bear no escape mutations at the last time point. (B) Part of the S-protein sequences from the indicated mice (arrows and numbers) are shown below as chromatograms, with mutations marked by arrowheads. (B) discloses the S-protein amino acid and nucleotide sequences as SEQ ID NOS 1477 and 1478, respectively. The sequences represented by the subsequent chromatograms that disclose amino acid residues and nucleotides are SEQ ID NOS 1479, 1480, 1480, 1480-1482, 1479, 1478, 1480, 1480, 1478, 1480, and 1483-1488, respectively, in order of columns. (C-D) HBsAg levels in mouse sera before and after antibody infusion. Mice were treated by anti-HBs combination H017+H019 (C) (see FIG. 7K) and H016+H017+H019 (D) (see FIG. 7L). Each line represents a mouse with concentrations of serum HBsAg level expressed in NCU/ml (national clinical units per milliliter).

DETAILED DESCRIPTION OF THE DISCLOSURE

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

This disclosure includes every nucleotide sequence described herein, and in the tables and figures, and all sequences that are complementary to them, RNA equivalents of DNA sequences, all amino acid sequences described herein, and all polynucleotide sequences encoding the amino acid sequences. Every antibody sequence and functional fragments of them are included. Polynucleotide and amino acid sequences having from 80-99% similarity, inclusive, and including ranges of numbers there between, with the sequences provided here are included in the invention. All of the amino acid sequences described herein can include amino acid substitutions, such as conservative substitutions, that do not adversely affect the function of the protein or polypeptide that comprises the amino acid sequences. It will be recognized that when reference herein is made to an “antibody” it does not necessarily mean a single antibody molecule. For example, “administering an antibody” includes administering a plurality of the same antibodies. Likewise, a composition comprising an “antibody” can comprise a plurality of the same antibodies.

For amino acid and polynucleotide sequences of this disclosure, contiguous segments of the sequences are included, and can range from 2 amino acids, up to full-length protein sequences. Polynucleotide sequences encoding such segments are also included.

The disclosure includes DNA and RNA sequences encoding the antibodies and antigen fragments thereof, and any virus peptides described herein for use in prophylactic and therapeutic approaches as protein or DNA and/or RNA vaccines, which may be formulated and/or delivered according to known approaches, given the benefit of this disclosure. The disclosure includes a cDNA sequences encoding the antibodies, antigen binding fragments thereof, and any viral proteins or peptides described herein. Expression vectors that contain cDNAs are also included, and encode said antibodies, antigen binding fragments thereof, and viral proteins and peptides.

All sequences from the figures, text, and tables of this application or patent include every amino acid sequence associated with every Donor ID, and all possible combinations of the amino acid sequences given for all complementarity determining regions (CDRs), e.g., all combinations of heavy chain CDR1, CDR2, CDR3 sequences, and all combinations of light chain CDR1, CDR2, and CDR3 sequences, including heavy chain sequences, and light chain sequences that are either lambda or kappa light chain sequences.

The disclosure includes all combinations of antibodies described herein. One or more antibodies may also be excluded from any combination of antibodies.

The disclosure includes antibodies described herein, which are present in an in vitro complex with one or more hepatitis B proteins.

In embodiments, the disclosure provides an isolated or recombinant antibody that binds with specificity to a hepatitis B virus epitope, and wherein the antibody optionally comprises a modification of its amino acid sequence, including but not limited to a modification of its constant region.

In embodiments, one or more antibodies described herein bind with specificity to an epitope present in the HBsAg protein or the S-protein in the unmutated, or mutated form.

In embodiments, the antibodies described herein bind to a hepatitis B protein that comprises one or more HepB escape mutations. In embodiments, the antibodies bind to a hepatitis B virus protein that comprises a mutation that is a substitution of a large positively charged residue for a small neutral residue. In embodiments, the mutation is present in the so-called “a” determinant area, which is known in the art. In embodiments, the epitope is present in the major hydrophilic region of the HBsAg protein. In embodiments, the epitope to which the antibodies bind is present in the S-protein, including but not necessarily limited to the predicted or actual extracellular domain of the S-protein.

In embodiments, the epitope to which the described antibodies bind is common to HBsAg L-protein, M-protein, or S-protein. In embodiments, the antibodies bind to an epitope present in the L-protein version of HBsAg, which comprises the amino acid sequence that is accessible via Accession number: AAL66340.1 as that amino acid sequence exists in the database as of the filing date of this application or patent. In an embodiment, this amino acid sequence is:

(SEQ ID NO: 2) MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKD HWPEANQVGAGAFGPGFTPPHGGLLGWSPQAQGILTTVPVAPPPASTNRQ SGRQPTPISPPLRDSHPQAMQWNSTTFHQALLDPRVRGLYFPAGGSSSGT VNPVPTTASPISSIFSRTGDPAPNMESTTSGFLGPLLVLQAGFFLLTRIL TIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMC LRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTSTTSTGPCKTCTS PAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLL VPFVQWFVGLSPTVWLSVIWMMWYWGPCLYNILSPFLPLLPIFFCLW VYI.

In embodiments, the disclosure includes use of only two proteins, or at least two proteins. In an embodiment, the S proteins may be used as bait to sort B cells purified from Chinese hamster ovary (CHO) cells, or any other suitable cell type, including but not limited to human cell cultures. In embodiments, the S protein comprises or consists of the amino acid sequence available under Uniprot ID P30019, the amino acid sequence of which is incorporated herein as it exists in the database at the filing date of this application or patent. In an embodiment, the S protein comprises the sequence:

(SEQ ID NO: 3) MENTASGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPG QNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDY HGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCI PIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWY WGPSLYNILSPFLPLLPIFFCLWVYI.

In non-limiting embodiments, the S polynucleotide sequence used for alanine scanning comprises:

(SEQ ID NO: 4) ATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGC GGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACT CGTGGTGGACTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGC CAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCC AATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATATTCC TCTTCATCCTGCTGCTATGCCTCATCTTCTTATTGGTTCTTCTGGATTAT CAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCAACAACAACCAG TACGGGACCATGCAAAACCTGCACGACTCCTGCTCAAGGCAACTCTATGT TTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAATTGCACCTGTATT CCCATCCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTC AGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCG TAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTAT TGGGGGCCAAGTCTGTACAGCATCGTGAGTCCCTTTATACCGCTGTTACC AATTTTCTTTTGTCTCTGGGTATACATTTAA.

The amino acid sequence encoded by the DNA sequence immediately above is:

(SEQ ID NO: 5) MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLG QNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDY QGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCI PIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWY WGPSLYSIVSPFIPLLPIFFCLWVYI.

In embodiments, antibodies of this disclosure bind to an epitope present in any of the foregoing amino sequences, including linear and confirmation epitopes that may be formed by proteins comprising or consisting of said sequences.

In an embodiment, the isolated or recombinant antibody or antigen binding fragment thereof binds with specificity to an epitope comprised by a structurally defined peptide loop, as further described herein. In embodiments, the loop is as generally depicted in FIG. 6, which comprises a partial structure of HepB surface protein, and demonstrates the existence of a loop that includes the most frequently targeted residue found in human escapes G145. Without intending to be bound by any particular theory, it is considered that this structure explains why this mutant can escape, and also why additional commonly found escape mutants exist. Further, the structure and the antibody peptide complex represents a new and previously undiscovered target for drug discovery. Thus, in embodiments, the disclosure provides for screening drug candidates that can interfere with formation of this structure, and thus which may also interfere with the viability of the virus. Those skilled in the art will recognize from the present disclosure how to design an assay to determine whether or not drug candidates could interfere with the complex, and how antibodies described in herein may be used in such an assay.

In embodiments, antibodies described herein bind with specificity to an amino acid sequence comprised by any peptide sequence described herein. In embodiments, the peptide comprises the sequence KPSDG (SEQ ID NO: 6), or mutants thereof. In embodiments, antibodies described herein bind with specificity to an epitope in an amino acid sequence that comprises the sequence PSSSSTKPSDGNSTS (SEQ ID NO: 7), or mutants thereof. Additional and non-limiting examples of peptides of this disclosure include those shown on FIG. 6, e.g., peptide-11 and peptide-12.

In embodiments, the disclosure comprises compositions and methods that involve use of more than one distinct antibody or antigen binding fragment thereof. In embodiments, the methods of this disclosure comprise administering a combination of antibodies or antigen binding fragment thereof which bind distinct hepatitis B epitopes. In embodiments, distinct antibodies recognize epitopes present in two dominant non-overlapping antigenic sites on the HBsAg, or epitopes present on the S-protein. In embodiments, the disclosure provides for use of a combination of the Group-I and Group-II antibodies described herein. Thus, the disclosure comprises co-administration or sequential administration of a combination of antibodies. In an embodiment, administration of a combination of distinct antibodies suppresses formation of viruses that are resistant to the effects of any one of the antibodies alone. In embodiments, the disclosure includes administering a combination comprising at least one Group I antibody and at least one Group II antibody, wherein at least one of the antibodies is G145R mutation resistant. In non-limiting embodiments, antibodies that are provided by the present disclosure, and which can be administered to an individual in need thereof, comprise at least one of H006, H007, H0017, H0019, or H020. Further, H005, H008 and H009 are similar to H006, and therefore may be used as alternatives to H006.

All combinations of H and L chains described herein are included, including all kappa and lambda light chains. In embodiments, a single antibody of this disclosure may comprise an H+L chain from one antibody, and an H+L chain from another antibody. In embodiments, the antibodies comprise modifications that are not coded for in any B cells obtained from an individual, and/or the antibodies are not produced by immune cells in an individual from which a biological sample from the individual is used at least in part to identify and/or generate and/or characterize the antibodies of this disclosure. In embodiments, antibodies provided by this disclosure can be made recombinantly, and can be expressed with a constant region of choice, which may be different from a constant region that was coded for in any sample from which the amino acid sequences of the antibodies were deduced.

As discussed above, in embodiments, the disclosure includes a combination of antibodies or antigen binding fragments thereof, or a composition comprising or consisting of said antibodies or antigen binding fragments thereof. In embodiments, a combination of antibodies of this disclosure are effective in preventing viral escape by mutation. In this regard, the disclosure includes data demonstrating that not all antibody combinations are effective in preventing escape by mutation, such as the combination of H006 and H007, which are ineffective. Thus, in embodiments, a combinations of antibodies or antigen binding fragments collectively target more than one commonly occurring escape mutation, examples of which escape mutations are known in the art and are described herein. Accordingly, combinations of antibodies and antigen binding fragments thereof of this disclosure may target non-overlapping groups of common escape mutations. In embodiments, the disclosure thus includes a proviso that excludes any combination of antibodies that collectively only target separate epitopes but have overlapping sensitivity to commonly occurring escape mutations.

In embodiments, at least one antibody or antigen binding fragment thereof included in this disclosure, and in the combinations and methods of this disclosure, has greater virus neutralizing activity than a control neutralizing activity value, such as the neutralizing capability of libivirumab. In embodiments, at least one antibody or antigen binding fragment of this disclosure exhibits a viral neutralizing activity with an IC₅₀ values that is less than 128 ng/ml, or less than 35 ng/ml, or less than 5 ng/ml, and including all integers and ranges of integers between 128 and 5 ng/ml. Such neutralizing activity can be determined using known approaches, such as by ELISA or immunofluorescence assays, and as further described in Example 5 of this disclosure. In embodiments, an antibody or antigen binding fragment thereof that is encompassed by this disclosure includes but is not limited to antibodies or antigen binding fragments selected from the H016, H017 and H019 antibodies, as defined by their CDRs. In an embodiment, the disclosure includes combinations of these antibodies, and can include antigen binding fragments thereof. In embodiments, the combination of antibodies comprises the H017 and H019 antibodies, and/or antigen binding fragments thereof. In an embodiment, the combination optionally further comprises the H016 antibody or an antigen binding fragment thereof. In embodiments, a combination of the disclosure comprises a combination that consists of only the H017 and H019 antibodies or antigen binding fragments thereof. In embodiments, a combination of the disclosure comprises a combination that consists of only the H016, H017, and H019 antibodies or antigen binding fragments thereof. Methods of administration of the described antibody combinations, and all other antibodies and antigen binding fragments thereof described herein, sequentially and concurrently are included within the scope of this disclosure. Thus, the disclosure includes administering to an individual in need concurrently or sequentially a combination of antibodies or antigen binding fragments thereof, which in certain embodiments comprise or consist of H017 and H019, or H016, H017, and H019 and antigen binding fragments thereof. Additional antibodies and antibody combinations, including antigen binding fragments thereof, include but are not limited to antibodies and antigen binding fragments thereof that comprise the heavy and light chain CDRs of H004, H005, and H009, and H020.

With respect to the H016, H017, and H019 antibodies, as can been seen from Table S2, the H016 antibody comprises a heavy chain CDR1 with the amino acid sequence GFTFPSHT (SEQ ID NO: 8), a heavy chain CDR2 with the amino acid sequence ISTTSEAI (SEQ ID NO: 9), and a heavy chain CDR3 with the amino acid sequence ARVGLALTISGYWYFDL (SEQ ID NO: 10). The H016 antibody comprises a kappa light chain CDR1 with the amino acid sequence QSISSN (SEQ ID NO: 11), a kappa light chain with the CDR2 amino acid sequence RAS, and a kappa light chain with the CDR3 amino acid sequence QQYDHWPLT (SEQ ID NO: 12).

As can be seen from Table S2, the H017 antibody comprises a heavy chain CDR1 with the amino acid sequence GFTFSNYW (SEQ ID NO: 13), a heavy chain CDR2 with the amino acid sequence ISTDGSST (SEQ ID NO: 14), and a heavy chain CDR3 with the amino acid sequence ARGSTYYFGSGSVDY (SEQ ID NO: 15). The H017 antibody comprises a lambda light chain with the CDR1 sequence SSDIGVYNY (SEQ ID NO: 16), a lambda light chain with the CDR2 sequence DVT, and a lambda light chain with the CDR3 sequence SSYRGSSTPYV (SEQ ID NO: 17).

As can be seen from Table S2, the H019 antibody comprises a heavy chain CDR1 with the amino acid sequence GGSITTGDYY (SEQ ID NO: 18), a heavy chain CDR2 with the amino acid sequence IYYSGST (SEQ ID NO: 19), and a heavy chain CDR3 with the amino acid sequence AIYMDEAWAFE (SEQ ID NO: 20). The H019 antibody comprises a lambda light chain CDR1 with the amino acid sequence QSIGNY (SEQ ID NO: 21), a lambda light chain with the CDR2 amino acid sequence AVS, and a lambda light chain with the CDR3 amino acid sequence QQSYTISLFT (SEQ ID NO: 22).

In certain embodiments, the antibodies contain one or more modifications, such as non-naturally occurring mutations. As non-limiting examples, in certain approaches the Fc region of the antibodies can be changed, and may be of any isotype, including but not limited to any IgG type, or an IgA type, etc. Antibodies of this disclosure can be modified to improve certain biological properties of the antibody, e.g., to improve stability, to modify effector functions, to improve or prevent interaction with cell-mediated immunity and transfer across tissues (placenta, blood-brain barrier, blood-testes barrier), and for improved recycling, half-life and other effects, such as manufacturability and delivery.

In embodiments, an antibody of this disclosure can be modified by using techniques known in the art, such as those described in Buchanan, et al., Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression mAbs 5:2, 255-262; March/April 2013, and in Zalevsky J. et al., (2010) Nature Biotechnology, Vol. 28, No. 2, p 157-159, and Ko, S-Y, et al., (2014) Nature, Vol. 514, p 642-647, and Horton, H. et al., Cancer Res 2008; 68: (19), Oct. 1, 2008, from which the descriptions are incorporated herein by reference. In certain embodiments an antibody modification increases in vivo half-life of the antibody (e.g. LS mutations), or alters the ability of the antibody to bind to Fc receptors (e.g. GRLR mutations), or alters the ability to cross the placenta or to cross the blood-brain barrier or to cross the blood-testes barrier. Thus, in certain embodiments an antibody modification comprises a change of G to R, L to R, M to L, or N to S, or any combination thereof.

In embodiments bi-specific antibodies are provided by modifying and/or combining segments of antibodies as described herein, such as by combining heavy and light chain pairs from distinct antibodies into a single antibody. Suitable methods of making bispecific antibodies are known in the art, such as in Kontermann, E. et al., Bispecific antibodies, Drug Discovery Today, Volume 20, Issue 7, July 2015, Pages 838-847, the description of which is incorporated herein by reference.

In embodiments, any antibody described herein comprises a modified heavy chain, a modified light chain, a modified constant region, or a combination thereof, thus rendering them distinct from antibodies produced by humans. In embodiments, the modification is made in a hypervariable region, and/or in a framework region (FR).

In embodiments, mutations to an antibody described herein, including but not limited to the antibodies described, comprise modifications relative to the antibodies originally produced in humans. Such modifications include but are not necessarily limited to the heavy chain to increase the antibody half-life.

In embodiments, antibodies of this disclosure have variable regions that are described herein, and may comprise or consist of any of these sequences, and may include sequences that have from 80-99% similarity, inclusive, and including ranges of numbers there between, with the sequences expressly disclosed herein, provided antibodies that have differing sequences retain the same or similar binding affinity as an antibody with an unmodified sequence. In embodiments, the sequences are at least 95%, 96%, 97%, 98% or 99% similar to an expressly disclosed sequence herein.

Antibodies comprising the sequences described in Table S2 have been isolated and characterized for at least binding affinity, and as otherwise described herein, such as for virus neutralizing activity. Thus, in embodiments the disclosure provides neutralizing antibodies. The term “neutralizing antibody” refers to an antibody or a plurality of antibodies that inhibits, reduces or completely prevents viral infection. Whether any particular antibody is a neutralizing antibody can be determined by in vitro assays described in the examples below, and as is otherwise known in the art. The term “broadly neutralizing” antibody refers to an antibody that can neutralize more than one strain or serotype of a virus.

Antibodies of this disclosure can be provided as intact immunoglobulins, or as antigen binding fragments of immunoglobulins, including but not necessarily limited to antigen-binding (Fab) fragments, Fab′ fragments, (Fab′)₂ fragments, Fd (N-terminal part of the heavy chain) fragments, Fv fragments (the two variable domains), dAb fragments, single domain fragments or single monomeric variable antibody domains, isolated CDR regions, single-chain variable fragment (scFv), and other antibody fragments that retain virus-binding capability and preferably virus neutralizing activity as further described below. In embodiments, the variable regions, including but not necessarily limited to the described CDRs, may be used as a component of a Bi-specific T-cell engager (BiTE), bispecific killer cell engager (BiKE), or a chimeric antigen receptor (CAR), such as for producing chimeric antigen receptor T cells (e.g. CAR T cells). In embodiments, the disclosure includes tri-valent antibodies, which can bind with specificity to three different epitopes.

Antibodies and antigens of this disclosure can be provided in pharmaceutical formulations. It is considered that administering a DNA or RNA polynucleotide encoding any protein described herein (including peptides and polypeptides), such as antibodies and antigens described herein, is also a method of delivering such proteins to an individual, provided the protein is expressed in the individual. Methods of delivering DNA and RNAs encoding proteins are known in the art and can be adapted to deliver the protein, particularly the described antigens, given the benefit of the present disclosure. Similarly, the antibodies of this disclosure can be administered as DNA molecules encoding for such antibodies using any suitable expression vector(s), or as RNA molecules encoding the antibodies.

Pharmaceutical formulations containing antibodies or viral antigens or polynucleotides encoding them can be prepared by mixing them with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents and the like. The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers. Examples of carriers include water, saline solutions or other buffers (such as phosphate, citrate buffers), oil, alcohol, proteins (such as serum albumin, gelatin), carbohydrates (such as monosaccharides, disaccharides, and other carbohydrates including glucose, sucrose, trehalose, mannose, mannitol, sorbitol or dextrins), gel, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, liposomes, antioxidants, chelating agents such as EDTA; salt forming counter-ions such as sodium; non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG), or combinations thereof. In embodiments, a pharmaceutical/vaccine formulation exhibits an improved activity relative to a control, such as antibodies that are delivered without adding additional agents, or a particular added agent improves the activity of the antibodies.

The formulation can contain more than one antibody type or antigen, and thus mixtures of antibodies, and mixtures of antigens, and combinations thereof as described herein can be included. These components can be combined with a carrier in any suitable manner, e.g., by admixture, solution, suspension, emulsification, encapsulation, absorption and the like, and can be made in formulations such as tablets, capsules, powder (including lyophilized powder), syrup, suspensions that are suitable for injections, ingestions, infusion, or the like. Sustained-release preparations can also be prepared.

The antibodies and vaccine components of this disclosure are employed for the treatment and/or prevention of hepatitis B virus infection in a subject, as well as for inhibition and/or prevention of their transmission from one individual to another.

The term “treatment” of viral infection refers to effective inhibition of the viral infection so as to delay the onset, slow down the progression, reduce viral load, and/or ameliorate the symptoms caused by the infection.

The term “prevention” of viral infection means the onset of the infection is delayed, and/or the incidence or likelihood of contracting the infection is reduced or eliminated.

In embodiments, to treat and/or prevent viral infection, a therapeutic amount of an antibody or antigen vaccine disclosed herein is administered to a subject in need thereof. The term “therapeutically effective amount” means the dose required to effect an inhibition of infection so as to treat and/or prevent the infection.

The dosage of an antibody or antigen vaccine depends on the disease state and other clinical factors, such as weight and condition of the subject, the subject's response to the therapy, the type of formulations and the route of administration. The precise dosage to be therapeutically effective and non-detrimental can be determined by those skilled in the art. As a general rule, a suitable dose of an antibody for the administration to adult humans parenterally is in the range of about 0.1 to 20 mg/kg of patient body weight per day, once a week, or even once a month, with the typical initial range used being in the range of about 2 to 10 mg/kg. Since the antibodies will eventually be cleared from the bloodstream, re-administration may be required. Alternatively, implantation or injection of the antibodies provided in a controlled release matrix can be employed.

The antibodies can be administered to the subject by standard routes, including oral, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular). In addition, the antibodies and/or the antigen vaccines can be introduced into the body, by injection or by surgical implantation or attachment such that a significant amount of an antibody or the vaccine is able to enter blood stream in a controlled release fashion. In certain embodiments antibodies described herein are incorporated into one or more prophylactic compositions or devices to, for instance, neutralize a virus before it enters cells of the recipient's body. For example, in certain embodiments a composition and/or device comprises a polymeric matrix that may be formed as a gel, and comprises at least one of hydrophilic polymers, hydrophobic polymers, poly(acrylic acids) (PAA), poly(lactic acids) (PLA), carageenans, polystyrene sulfonate, polyamides, polyethylene oxides, cellulose, poly(vinylpyrrolidone) (PVP), poly(vinyl alcohol) (PVA), chitosan, poly(ethylacrylate), methylmethacrylate, chlorotrimethyl ammonium methylmethacrylate, hydroxyapatite, pectin, porcine gastric mucin, poly(sebacic acid) (PSA), hydroxypropyl methylcellulose (HPMC), cellulose acetate phthalate (CAP), magnesium stearate (MS), polyethylene glycol, gum-based polymers and variants thereof, poly (D,L)-lactide (PDLL), polyvinyl acetate and povidone, carboxypolymethylene, and derivatives thereof. In certain aspects the disclosure comprises including antibodies in micro- or nano-particles formed from any suitable biocompatible material, including but not necessarily limited to poly(lactic-co-glycolic acid) (PLGA). Liposomal and microsomal compositions are also included. In certain aspects a gel of this disclosure comprises a carbomer, methylparaben, propylparaben, propylene glycol, sodium carboxymethylcellulose, sorbic acid, dimethicone, a sorbitol solution, or a combination thereof. In embodiments a gel of this disclosure comprises one or a combination of benzoic acid, BHA, mineral oil, peglicol 5 oleate, pegoxol 7 stearate, and purified water, and can include any combination of these compositions.

Antibodies of this disclosure can be produced by utilizing techniques available to those skilled in the art. For example, one or distinct DNA molecules encoding one or both of the H and L chains of the antibodies can be constructed based on the coding sequence using standard molecular cloning techniques. The resulting DNAs can be placed into a variety of suitable expression vectors known in the art, which are then transfected into host cells, which are preferably human cells cultured in vitro, but may include E. coli or yeast cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, and human embryonic kidney 293 cells, etc. Antibodies can be produced from a single, or separate expression vectors, including but not limited to separate vectors for heavy and light chains, and may include separate vectors for kappa and lambda light chains as appropriate.

In embodiments, the antibodies may be isolated from cells. In embodiments, the antibodies are recombinant antibodies. “Recombinant” antibodies mean the antibodies are produced by expression within cells from one or more expression vectors.

In certain approaches the disclosure includes neutralizing antibodies as discussed above, and methods of stimulating the production of such antibodies.

In certain approaches the disclosure includes vaccinating an individual using a composition described herein, and determining the presence, absence, and/or an amount of neutralizing antibodies produced in response to the vaccination. Thus, methods of determining and monitoring efficacy of a vaccination at least in terms of neutralizing antibody production are included. In an embodiment, subsequent to determining an absence of neutralizing antibodies, and/or an amount of neutralizing antibodies below a suitable reference value, the invention includes administering a composition disclosed herein to the individual. Subsequent administrations and measurements can be made to track the treatment efficacy and make further adjustments to treatment accordingly.

Antibodies and proteins of this disclosure can be detectably labeled and/or attached to a substrate. Any substrate and detectable label conventionally used in immunological assays and/or devices is included. In embodiments the substrate comprises biotin, or a similar agent that binds specifically with another binding partner to facilitate immobilization and/or detection and/or quantification of antibodies and/or viral proteins.

In embodiments any type of enzyme-linked immunosorbent (ELISA) assay can be used, and can be performed using polypeptides and/or antibodies of this disclosure for diagnostic purposes, and can include direct, indirect, and competitive ELISA assays, and adaptations thereof that will be apparent to those skilled in the art given the benefit of this disclosure.

Any diagnostic result described herein can be compared to any suitable control. Further, any diagnostic result can be fixed in a tangible medium of expression and communicated to a health care provider, or any other recipient. In one aspect the disclosure comprises diagnosing an individual as infected with hepatitis B virus and administering a composition of this invention to the individual.

In certain embodiments the disclosure includes one or more recombinant expression vectors encoding at least H and L chains of an antibody or antigen binding fragment of this disclosure, cells and cell cultures comprising the expression vectors, methods comprising culturing such cells and separating antibodies from the cell culture, the cell culture media that comprises the antibodies, antibodies that are separated from the cell culture, and kits comprising the expression vectors encoding an antibody and/or a polypeptide of this disclosure. Products containing the antibodies and/or the polypeptides are provided, wherein the antibodies and/or the polypeptides are provided as a pharmaceutical formulation contained in one or more sealed containers, which may be sterile and arranged in any manner by which such agents would be suitable for administration to a human or non-human subject. The products/kits may further comprise one or more articles for use in administering the compositions.

The following Examples are intended to illustrate but not limit the invention.

Example 1

Serologic Responses Against HBV

To select individuals with outstanding antibody responses to HBsAg, we performed ELISA assays on serum obtained from 159 volunteers. These included 15 uninfected and unvaccinated controls (HBsAg⁻, anti-HBs⁻, anti-HBc⁻), 20 infected and spontaneously recovered (HBsAg⁻, anti-HBs^(+/−), anti-HBO, and 124 vaccinated (HBsAg⁻, anti-HBs^(+/−), anti-HBc⁻) volunteers. These individuals displayed a broad spectrum of anti-HBs titers (x-axis in FIG. 1A and FIG. 8B; Table S1). To determine their neutralizing activity, we tested their ability to block HBV infection in sodium taurocholate co-transporting polypeptide (NTCP)-overexpressing HepG2 cells (Michailidis et al., 2017; Yan et al., 2012) (y-axis in FIG. 1A and FIGS. 8B and 8C; Table S1). Sera or antibodies purified from individuals with high levels of neutralizing activity were then compared across a wide range of dilutions (FIGS. 1B and 1C). Although anti-HBs ELISA titers positively correlated with neutralizing activity (r_(s)=0.492, p<0.001, Spearman's rank correlation), there were notable exceptions as exemplified by volunteers #99 and #49, whose sera failed to neutralize HBV despite high anti-HBs ELISA titers (FIG. 1A). Thus, ELISA titers against HBsAg are not entirely predictive of neutralizing activity in vitro.

The HBV surface protein, HBsAg can be subdivided into PreS1-, PreS2- and S-regions (FIG. 1D). To determine which of these regions is the dominant target of the neutralizing response in the selected top neutralizers, we used S-protein to block neutralizing activity in vitro. The neutralizing activity in volunteers that received the HBV vaccine, which is composed of S-protein, was completely blocked by S-protein (black lines in FIG. 1E). The same was true for the spontaneously recovered individuals in our cohort despite a reported ability of this population to produce anti-PreS1 or anti-PreS2 antibodies (Coursaget et al., 1988; Li et al., 2017; Sankhyan et al., 2016) (red lines in FIG. 1E). These results suggest that the neutralizing antibody response in the selected individuals is directed primarily against the S-protein irrespective of immunization or infection.

Example 2

Human Monoclonal Antibodies to HBV

To characterize the antibodies responsible for neutralizing activity in the selected individuals, we purified S-protein binding class-switched memory B cells (Escolano et al., 2019; Scheid et al., 2009a). Unexposed naïve controls and vaccinated individuals with low anti-HBs ELISA titers showed background levels of S-protein specific memory B cells (FIGS. 2A and 9A). In contrast, individuals with high neutralizing activity displayed a distinct population of S-antigen binding B cells constituting 0.03-0.07% of the IgG⁺ memory compartment (CD19-MicroBeads⁺ CD20-PECy7⁺ IgG-Bv421⁺ S-protein-PE⁺ S-protein-APC⁺ ovalbumin-Alexa Fluor 488⁻) (FIGS. 2A and 9A). Consistent with the findings in elite HIV-1 neutralizers (Rouers et al., 2017), the fraction of S-protein specific cells was directly correlated to the neutralization titer of the individual (r_(s)=0.699, p=0.0145, Spearman's rank correlation) (FIG. 2B).

Immunoglobulin heavy (IGH) and light (IGL or IGK) chain genes were amplified from single memory B cells by PCR (Robbiani et al., 2017; Scheid et al., 2009b; von Boehmer et al., 2016). Overall, we obtained 244 paired heavy and light chain variable regions from S-protein-binding memory B cells from eight volunteers with high anti-HBs ELISA titers (FIGS. 9B and 9C; Table S2). Expanded clones composed of cells producing antibodies encoded by the same Ig variable gene segments with closely related CDR3s were found in each of the top neutralizers #146, #60 and #13 (FIG. 2C). Moreover, IGHV3-30/IGLV3-21 was present in #146 and #60; IGHV3-33/IGLV3-21 in #146 and #13; and IGHV3-23/IGLV3-21 in #146, #60 and #13. The variable diversity and joining (V(D)J) region of these antibodies was approximately 80% identical at the amino acid level (FIG. 2D). Antibodies with related Ig heavy and light chains were also identified between volunteer #55 (HBV infected but recovered) and vaccinated individuals (FIGS. 2C and 9B). We conclude that top HBV neutralizers produce clones of antigen-binding B cells that express related Ig heavy and light chains.

Example 3

Breadth of Reactivity

Twenty representative antibodies from 5 individuals, designated as H001 to H020, were selected for expression and further testing (FIG. 9B). All 20 antibodies showed reactivity to the S-protein antigen used for B cell selection (HBsAg adr CHO) by ELISA with 50% effective concentration (EC₅₀) values ranging from 18-350 ng/ml (FIG. 3A). These antibodies carried somatic mutations that enhanced antigen binding as determined by reversion to the inferred unmutated common ancestor (UCA) (FIG. 3B). Thus, affinity maturation was essential for their high binding activity.

Four major serotypes of HBV exist as defined by a constant “a” determinant and two variable and mutually exclusive determinants “d/y” and “w/r” (Bancroft et al., 1972; Le Bouvier, 1971) with a highly statistically significant association between serotypes and genotypes (Kramvis et al., 2008; Norder et al., 2004). To determine whether our antibodies cross-react to different HBsAg serotypes, we performed ELISAs with 5 additional HBsAg antigens: yeast-expressed serotype “adr”, “adw”, and “ayw”, as well as “ad” and “ay” antigen purified from human blood (FIG. 3C). Many of the antibodies tested displayed broad cross-reactivity and EC₅₀ values lower than libivirumab, a human anti-HBs monoclonal antibody that was isolated from HBV-immunized humanized mice and then tested clinically (Eren et al., 2000; Eren et al., 1998; Galun et al., 2002). These antibodies were not polyreactive or autoreactive when tested in polyreactivity ELISA and HEp-2 immunofluorescence assays respectively (FIGS. 10A and 10B). We conclude that the antibodies tested are broadly cross-reactive with different HBV serotypes.

Example 4

Antigenic Epitopes on S-Protein

To determine whether the selected antibodies bind to overlapping or non-overlapping epitopes, we performed competition ELISA assays, in which the S-protein was pre-incubated with a selected antibody followed by a second biotinylated antibody. Antibodies that showed weak levels of binding in ELISA (H002, H012, H013, H014, H018) were excluded. As expected, all of the antibodies tested blocked the binding of the autologous biotinylated monoclonal (yellow boxes in FIG. 4A), while control human anti-HIV antibody 10-1074 failed to block any of the anti-HBs antibodies. The competition ELISA identified three mutually exclusive groups of monoclonal antibodies, suggesting that there are at least three dominant non-overlapping antigenic sites on HBsAg (red box for Group-I, blue box for Group-II, and H017 in Group-III, FIG. 4A). Each of the individuals that had 2 or more antibodies tested in the competition ELISA expressed monoclonal antibodies that targeted 2 of the 3 non-overlapping epitopes (FIGS. 4A and 9B).

To further define these epitopes, we produced a series of alanine mutants spanning most of the predicted extracellular domain of the S-protein with the exception of cysteines, alanines, and amino acid residues critical for S-protein production (Salisse and Sureau, 2009) (FIG. 1D). ELISA assays with the representative antibodies from each antibody group and the mutant proteins revealed a series of binding patterns partially corresponding to the three groups defined in the competition assays (FIGS. 4B and 11). For example, mutations I110A and T148A interfered with binding by Group-I antibodies exemplified by H004, H006, H019, and H020, but had little measurable effect on Group-II antibodies exemplified by H007, H015, and H016 or Group-III antibody H017 (FIGS. 4B and 11).

However alanine scanning suggested that some residues such as D144 and G145 are critical for binding of monoclonals in both Group-I and Group-II despite their inability to compete with each other for binding to the native antigen (FIGS. 4B and 11). Without intending to be constrained by any particular theory, it is considered that D144A and G145A mutations alter the overall structure of HBsAg thereby interfering with binding of antibodies that normally target independent sites on the protein.

In addition to alanine scanning, we also produced 44 common naturally occurring escape variants found in chronically infected individuals (Hsu et al., 2015; Ijaz et al., 2012; Ma and Wang, 2012; Salpini et al., 2015). Whereas alanine scanning showed that some of the antibodies in Group-I and -II were resistant to G145A, the corresponding naturally occurring mutations at the same position, G145E and G145R, revealed decreased binding by most antibodies (FIG. 4C). Among the antibodies tested, H017 and H019, in Groups-I and -III respectively, showed the greatest resistance to G145 mutations and the greatest breadth and complementarity (FIG. 4C). We conclude that human anti-HBs monoclonals obtained from the selected individuals recognize distinct epitopes on HBsAg, most of which appear to be non-linear conformational epitopes spanning different regions of the protein.

Example 5

In Vitro Neutralizing Activity

To determine whether the new monoclonals neutralize HBV in vitro, we performed neutralization assays using HepG2-NTCP cells (FIGS. 5A and 5B). The 50% inhibitory concentration (IC₅₀) values were calculated based on HBsAg/HBeAg ELISA or immunofluorescence staining for HBcAg expression (Michailidis et al., 2017) (FIG. 5C). Neutralizing activity was further verified by in vitro neutralization assays using primary human hepatocytes (Michailidis et al., 2020) (FIGS. 5C and 5D). Fourteen of the 20 antibodies tested showed neutralizing activity with IC₅₀ values as low as 5 ng/ml (FIG. 5C). By comparison, libivirumab had an IC₅₀ of 35 and 128 ng/ml in the neutralization assays based on ELISA and immunofluorescence assays respectively (FIG. 5C). Somatic mutations were essential for potent neutralizing activity as illustrated by the reduced activity of the inferred UCAs (FIGS. 12A and 12B). In addition, optimal activity required bivalent binding since the IC₅₀ values for Fab fragments were 2 orders of magnitude higher than intact antibodies (FIG. 5E). Finally, there was no overt synergy when Group-I, -II, and -III antibodies were combined (FIG. 12C). We conclude that half of the new monoclonals were significantly more potent than libivirumab including Group-I H004, H005, H006, H008, H009, H019, and H020 and Group-II H007, H015, and H016 (FIG. 5C).

Example 6

Structure of the H015 Antibody/Peptide Complex

H015 differed from other antibodies in that its binding was inhibited by 5 consecutive alanine mutations spanning positions K141-G145 indicating the existence of a linear epitope. This idea was verified by ELISA against a series of overlapping peptides comprising the predicted extracellular domain of S-protein (FIGS. 6A and 13A). The data showed that H015 binds to KPSDGN (SEQ ID NO: 23), which is near the center of the putative extracellular domain and contains some of the most frequently mutated amino acids during natural infection.

To examine the molecular basis for H015 binding, its Fab fragment was co-crystallized with the target peptide epitope PSSSSTKPSDGNSTS (SEQ ID NO: 24), where all cysteine residues that flank the recognition sequence were substituted with serine to avoid non-physiological cross-linking. The 1.78 Å structure (FIGS. 6B and 13B) revealed that the peptide is primarily bound to the immunoglobulin heavy chain (FIGS. 6B and 6C), interacting with residues from CDR1 (R31), CDR2 (W52, F53) and CDR3 (E99, P101, L103, L104) of IgH with only one contact with CDR3 (P95) of IgL. The peptide adopts a three-residue beta hairpin (class 3) of the 3:5 type involving residues K141 through G145 as only one hydrogen bond is seen, between K141 and G145 (Milner-White and Poet, 1986), and they are not part of a beta sheet. The peptide is further stabilized by a salt-bridge formed between K141 and D144 (FIGS. 6D and 13C). Interestingly, the distance between the Cαs of the two residues (C139 and C147) flanking the recognition residues is 6.4 Å and are poised to form a disulfide bond between C139 and C147 found in the native HBsAg structure (Ito et al., 2010). The H015 Fab appears to stabilize the conformation of the peptide via the Fab-peptide contacts (FIG. 13D) including a large binding surface (866 Å²; antibody-antigen buried surface of 600-900 Å² (Braden and Poljak, 1995)) comprised primarily of a single salt-bridge (lysine to aspartate; 0.9±0.3 Kcal/mol) (White et al., 2013) and five hydrogen bonds (1-2 Kcal/mol/bond) (Sheu et al., 2003). Moreover, the peptide further restricts loop through intra-peptide contacts (FIG. 13D) even in the absence of the disulfides.

The residues that form the hairpin are important for anti-HBs antibody recognition as determined by alanine scanning (FIGS. 4B and 11). In addition, each of these residues has been identified as important for immune recognition during natural infection (Ma and Wang, 2012). G145R, the most common naturally occurring S-protein escape mutation substitutes a large positively charged residue for a small neutral residue (circled residue in FIG. 6E) potentially altering the antigenic binding surface. G145 adopts a positive phi angle of 77.9 and by doing so introduces a kink in the beta-strand, a structure that would be disrupted with the substitution to arginine.

HBsAg can be glycosylated at N146 and this site is also strictly conserved. However, some studies have suggested that this glycosylation site is never fully occupied, resulting in a nearly 1:1 ratio of glycosylated and non-glycosylated isoforms on the surface of viral envelope (Julithe et al., 2014). The glycosylation may be either NAG-NAG-MAN or NAG-(FUC)-NAG-MAN (Hyakumura et al., 2015). We have modeled both fucosylated and non-fucosylated options by grafting a 7mer and 11mer glycan conjugated at N146 of peptide in the presence of the Fab. We found that both glycosylation forms are tolerated at that location with only minimal torsional adaptations without clashes with the Fab, though the fucosylated (branched) glycan required some additional torsional angle changes to the Fab, as well.

Example 7

Protection and Therapy in Humanized Mice

HBV infection is limited to humans, chimpanzees, tree shrews, and human liver chimeric mice (Sun and Li, 2017). To determine whether our anti-HBs bNAbs prevent infection in vivo we produced human liver chimeric Fah^(−/−) NODRag1^(−/−) IL2rg^(null) (huFNRG) mice (de Jong et al., 2014) and injected them with control or H020 (Group-I) or H007 (Group-II) antibodies before infection with HBV (FIG. 7A-7D). These two antibodies were chosen because they bind to non-overlapping sites, and have broad and potent neutralizing activity. Whereas all six control animals in two independent experiments were infected, pre-exposure prophylaxis with either H007 or H020 was fully protective (FIG. 7B-7D). We conclude that single anti-HBs bNAbs targeting different epitopes on the major virus surface antigen can prevent infection in vivo.

To determine whether bNAbs can also control established infections, we infused control antibody or bNAb H020 (Group-I) or H007 (Group-II) into huFNRG mice with HBV viral loads of 10⁶-10⁸ copies/ml of serum (FIG. 7E-7H and FIG. 14A). Fah^(−/−) NODRag1^(−/−) IL2rg^(null) mice are highly immunodeficient and unable to mount adaptive immune responses due to absence of T and B lymphocytes. In addition, the IL2rg^(null) mutation prevents cytokine signaling through multiple receptors, leading to a deficiency in innate immune function including antibody-dependent cellular cytotoxicity. Thus, elimination of viremia of 10⁶-10⁸ DNA copies/ml in huFNRG mice by antibody therapy alone would not be expected.

Animals that received the control antibodies further increased viremia to as high as ˜10¹¹ DNA copies/ml (FIG. 7F). In contrast, the 5 mice that received H020 maintained stable levels of viremia for around 30 days (FIG. 7G), after which time 2 mice showed increased viremia (arrow-1/3 in FIG. 7G). A similar result was observed in the 5 mice that received H007 (FIG. 7H), where only one showed a slight increase viremia at around day 50 (arrow-5 in FIG. 7H).

To determine whether the animals that showed increased HBV DNA levels during antibody monotherapy developed escape mutations, we sequenced the viral DNA recovered from mouse blood. All three mice that escaped H020 (Group-I) or H007 (Group-II) monotherapy developed viruses that carried a G145R mutation in the S-protein (arrow-1/3 in FIG. 7G, arrow-5 in FIG. 7H, FIG. 7I, and FIG. 14). This mutation represents a major immune escape mutation in humans (Zanetti et al., 1988). Furthermore, mutations at the same position in the S-protein were also identified in mice that maintained low level viremia (arrow-2/4 in FIG. 7G, arrow-6/7 in FIG. 7H, FIG. 7I, and FIG. 14), but not in control animals (FIG. 14). These results show that anti-HBs bNAb monotherapy leads to the emergence of escape mutations that are consistent with bNAb binding properties in vitro (FIG. 4C).

To determine whether a combination of bNAbs targeting 2 separate epitopes would interfere with the emergence of resistant strains, we co-administered H006+H007 (Group-I and -II, respectively) to 8 HBV-infected huFNRG mice (FIG. 7J). H006 (Group-I) was chosen for this purpose because of its resistance to D144A and G145A mutation (FIG. 4B). Similar to H007 monotherapy, there was only a slight increase in viremia in animals treated with the H006+H007 anti-HBs bNAb combination during the 60-day observation period (FIGS. 7J and 14A). However, sequence analysis revealed that 3 of the mice developed resistance mutations including K122R/G145R, C137Y, and C137Y/D144V (arrow-8/9/10 in FIG. 7J, FIG. 7I, and FIG. 14). These mutations confer loss of binding to both H006 (Group-I) and H007 (Group-II) (FIG. 4C). Thus, the combination of 2 anti-HBs bNAbs targeting separate epitopes but susceptible to the same clinical escape variants is not sufficient to inhibit emergence of escape mutations.

To attempt to block the emergence of escape mutations, we combined H017+H019 (Group-III and -I, respectively) bNAbs because they displayed complementary sensitivity to commonly occurring natural mutations (FIG. 4C). None of 7 mice treated with the combination of showed increased viremia or escape mutations as assessed by sequence analysis (FIGS. 7K and 14A). Similar effects were also observed in the 9 animals treated with the H016, H017 and H019 (Group-II, -III, -I, respectively) triple antibody combination (FIGS. 7L and 14A). Moreover, both these combinations dramatically reduced the HBsAg levels in serum (FIGS. 14C and 14D). Altogether, these findings suggest that control of HBV infection by bNAbs requires a combination of antibodies targeting non-overlapping groups of common escape mutations.

RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Experimental Models: Cell Lines Human Hepatocytes, Cryopreserved, Plateable and Interaction Qualified Lonza Bioscience Cat#HUCPI Hepatocyte Defined Medium Corning Cat#05449 HepG2-NTCP (Michailidis et al., 2017) N/A HEK293-6E National Research Council of Canada NRCfile 11565 HepDE19 cells (Cai et al., 2012) N/A Huh-7.5 (Robbiani et al., 2017) N/A Experimental Models: Mouse Strains Fah^(−/−)NODRag1^(−/−)IL2rg^(−/−) mouse (huFNRG) (de Jong et al., 2014) N/A Bacteria and Viruses Subcloning Efficiency ™ DH5α ™ Competent Cells Thermo Fisher Scientific Cat#18265017 HBV viruses (Cai et al., 2012) N Antibodies Human recombinant 10-1074 (Mouquet et al., 2012) N/A Human recombinant ED38 (Wardemann et al., 2003) N/A Human recombinant mG053 (Yurasov et al., 2005) N/A Goat anti-Human IgG (H + L) Secondary Antibody, HRP Thermo Fisher Scientific Cat#31410 Alexa Fluor 488 Mouse anti-Human CD19 BD Biosciences Cat#557697 BV421 Mouse Anti-Human CD 19 BD Biosciences Cat#562440 anti-CD20-PECy7 BD Biosciences Cat#335811 Anti-CD27-PE BD Biosciences Cat#555441 APC Mouse Anti-Human IgG BD Pharmingen Cat#550931 Bv421 Mouse Anti-Human IgG BD Biosciences Cat#562581 Anti-Hepatitis B virus core antigen IgG AUSTRAL Biologicals Cat#HBP-023-9 Alexa Fluor ® 488 AffiniPure Goat Anti-Human IgG, F(ab')2 fragment specific Jackson ImmunoResearch Cat#109-545-006 Goat anti-Rabbit IgG (H + L) Alexa Fluor 594 Thermo Fisher Scientific Cat#A11037 Chemicals and Proteins Streptavidin HRP BD Biosciences Cat#554066 APC Streptavidin BD Biosciences Cat#554067 Strep-PE eBioscience Cat# 12-4317-87 Streptavidin, Alexa Fluor ™ 488 Thermo Fisher Scientific Cat#S32354 Human BD Fc Block ™ BD Biosciences Cat#564220 Ovalbumin (257-264) chicken Sigma-Aldrich Cat#S7951 HBsAg adr CHO ProSpec Cat#HBS-875 HBsAg adw ProSpec Cat#HBS-872 HBsAg protein adr Fitzgerald Cat#30-AH37 HBsAg protein ay Fitzgerald Cat#30-1816 HBsAg protein ad Fitzgerald Cat#30-AH15 HBsAg protein ayw Fitzgerald Cat#30R-AH018 RNAsin Plus RNAse inhibitor Promega Cat#N2615 Random Primers Thermo Fisher Scientific Cat#48190011 Lipopolysaccharides from E. coli O55:B5 Sigma-Aldrich Cat#L2637 Insulin solution human Sigma-Aldrich Cat#I1927 Deoxyribonucleic acid from calf thymus Sigma-Aldrich Cat#4522 Hemocyanin from Megathura crenulata (keyhole limpet) Sigma-Aldrich Cat#H8283 Poly(ethylene glycol) Sigma-Aldrich Cat#81268 Normal Goat Serum Jackson ImmunoResearch Cat#005-000-121 DAPI, FluoroPure ™ grade Thermo Fisher Scientific Cat#D21490 Paraformaldehyde 4% Aqueous Solution Electron Microscopy Sciences Cat#157-4 Phusion High-Fidelity DNA Polymerase Thermo Fisher Scientific Cat#F-530L Commercial Assays ARCHITECT Anti-HBs Abbott Laboratories Cat#B7C180 ARCHITECT HBsAg Qualitative Abbott Laboratories Cat#BlP970 ARCHITECT Anti-HBc II Abbott Laboratories Cat#B8L440 HBsAg CLIA kit Autobio Diagnostics Co. Cat#CL0310-2 HBeAg CLIA kit Autobio Diagnostics Co. Cat#CL0312-2 Anti-HBs CLIA kit Autobio Diagnostics Co. Cat#CL0311-2 LS magnetic columns Miltenyi Biotech Cat#130-042-401 CD 19 MicroBeads, human Miltenyi Biotech Cat#130-097-05 5 EZ-Link ™ Micro NHS-PEG4- Biotinylation Kit Thermo Fisher Scientific Cat#21955 Superscript III Reverse Transcriptase Thermo Fisher Scientific Cat# 18080044 QIAamp DNA blood mini kit Qiagen Cat#51104 TaqMan Universal PCR Master Mix Applied Biosystems Cat#4304437 Antinuclear antibodies (HEp-2) Kit MBL International Cat#ANK-120 Centricon Plus-70 Ultracel PL-100 Millipore Sigma Cat#UFC710008 X-tremeGENE 9 DNA Transfection Reagent Sigma-Aldrich Cat#6365787001 Plasmids IGγ1 expression vector (von Boehmer et al., 2016) N/A IGκ expression vector (von Boehmer et al., 2016) N/A IGλ expression vector (von Boehmer et al., 2016) N/A p1.3xHBV-WT Laboratory of Charles M. Rice N/A Softwares and Websites PRISM GraphPad www.graphpad.com IgBlast (Ye et al., 2013) www.ncbi.nlm.nih.gov/igblast/ IMGT/V-QUEST (Lefranc et al., 2015) www.imgt.org/IMGT_vquest/vquest Geneious Prime Geneious www.geneious.com/

Example 8

This Example provides a description of materials, methods, and subjects used to obtain the foregoing results.

Experimental Models and Subjects

Human Subjects

Volunteer recruitment and blood draws were performed at the Rockefeller University Hospital under a protocol approved by the institutional review board (IRB QWA-0947). Study participants ranged in age from 22-65 with a mean of 43, the female:male ratio was 81:78 (FIGS. 8D and 8E; Table 51).

Animals

Fah^(−/−) NODRag1^(−/−) IL2rg^(null) (FNRG) female mice were produced as reported (de Jong et al., 2014) and maintained in the AAALAC-certified facility of the Rockefeller University. Animal protocols were in accordance with NIH guidelines and approved by the Rockefeller University Institutional Animal Care and Use Committee under protocol #18063. Female littermates were randomly assigned to experimental groups.

Cell Lines

HepG2-NTCP cells (Michailidis et al., 2017) and HepDE19 cells (Cai et al., 2012) were maintained in collagen-coated flasks in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% or 3% fetal bovine serum (FBS) and 0.1 mM non-essential amino acids (NEAA). Huh7.5-NTCP cells were maintained in DMEM supplemented with 10% FBS and 0.1 mM NEAA. All liver cell lines were cultured at 37° C. in 5% CO₂. Human embryonic kidney HEK293-6E suspension cells were cultured at 37° C. in 8% CO₂ with shaking at 120 rpm.

Viruses

HBV-containing supernatant from HepDE19 cells was collected and concentrated as previously described (Michailidis et al., 2017). The concentrated virus stock was aliquoted and stored at −80° C. For in vivo experiments one aliquot of mouse-passaged genotype C HBV virus, originally launched from patient serum (Billerbeck et al., 2016), was stored at −80° C. and thawed for mouse infection experiments. For protection and treatment experiments, animals were challenged intravenously using 1×10⁴ DNA copies per mouse.

Bacteria

E. coli DH5-alpha were cultured at 37° C. with shaking at 230 rpm.

Methods

Collection of Human Samples

Samples of peripheral blood were collected from volunteers at the Rockefeller University Hospital. Serum was isolated by centrifugation of coagulated whole blood, and aliquoted for storage at −80° C. PBMCs were isolated using a cell separation tube with frit barrier and cryopreserved in liquid nitrogen in 90% heat-inactivated FBS supplemented with 10% dimethylsulfoxide (DMSO).

HBV Stock

HepDE19 cells (Cai et al., 2012) were cultured in the absence of tetracycline to induce HBV replication. After seven days, supernatant was collected every other day for two weeks and fresh medium was added. After each collection, medium was spun down to remove cell debris, passed through a 0.22 μm filter, and kept at 4° C. Collected medium was concentrated 100-fold via centrifugation using Centricon Plus-70 centrifugal filter devices (Millipore-Sigma, Billerica, Mass.). Mouse-passaged genotype C HBV virus (Billerbeck et al., 2016) was used for in vivo mouse experiment.

In Vitro HBV Neutralization Assay

In vitro HBV infection was performed as previously described (Michailidis et al., 2017). Briefly, HepG2-NTCP cells were seeded in 96-well collagen-coated plates in DMEM supplemented with 10% FBS and 0.1 mM NEAA. The medium was changed to DMEM with 3% FBS, 0.1 mM NEAA, and 2% DMSO the next day and cultured for an additional 24 hours before infection. The inoculation was in DMEM supplemented with 3% FBS and 0.1 mM NEAA 4% PEG and 2% DMSO. Antibodies or serum samples were incubated with the virus in the inoculation medium for one hour at 37° C. before adding to cells. Serum neutralization capacity (y-axis in FIGS. 1A and 8B) was calculated as the reciprocal of the relative percentage of infected HepG2-NTCP cells immunostained by rabbit anti-HBV core antibody (AUSTRAL Biologicals). For example, if the relative percentage of infected cells were 100% (no serum added or the sera from unexposed naïve control donors), the serum neutralization capacity would be calculated as 1; but if the relative percentage of infected cells were 50% or 10%, the serum neutralization capacity would be 2 or 10. For the blocking neutralization assay, S-protein antigen at different concentration was incubated with purified polyclonal antibodies for one hour at 37° C. before incubation with HBV virus. The cells were then spinoculated for one hour by centrifugation at 1,000 g at 37° C. After a 24-hour incubation, supernatant was removed, cells were washed five times with PBS, and 100 μl of fresh DMEM supplemented with 3% FBS, 0.1 mM NEAA, and 2% DMSO. Both supernatant and cells were harvested 7 days after infection for analysis. Neutralization assays in primary human hepatocytes were performed as above using hepatocytes from livers of highly humanized mice that were harvested and seeded on collagen-coated plates in hepatocyte defined medium (Corning) (Michailidis et al., 2020).

Chemiluminescence Immunoassay

For quantitative analysis of secreted antigen HBsAg or HBeAg, 50 μl of the collected supernatant was loaded into 96-well plates of a chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou, China) according to the manufacturer's instructions. Plates were read using a FLUOstar Omega luminometer (BMG Labtech). The absolute concentrations were measured and the relative values were calculated by normalizing to the virus-only control well in the same lane. For example, the absolute HBsAg/HBeAg level in virus-only control well (considered as reference) was 20 NCU/ml (national clinical units per milliliter), while adding one neutralizing serum sample might reduce this to 5 NCU/ml. Therefore, after normalization, the relative HBsAg/HBeAg level were calculated as 100% in control and 25% for this neutralizing serum. Since many factors (virus concentration, cell concentration, immunofluorescence reading, etc.) vary between different plates or different rounds of experiments, normalization is necessary for combining data for comparison.

Immunofluorescence

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, washed with PBS and permeabilized with 0.1% Triton X-100 in PBS. After blocking with 5% goat serum, the cells were incubated with rabbit anti-HBV core antibody (AUSTRAL Biologicals) overnight at 4° C. and visualized with goat anti-rabbit Alexa Fluor 594 (Thermo Fisher Scientific). Nuclei were stained with DAPI. Cells were imaged using a Nikon Eclipse TE300 fluorescent microscope and processed with ImageJ. For high-content imaging analysis ImageXpress Micro XLS (Molecular Devices, Sunnyvale, Calif.) was used. The absolute HBc⁺ percentages were obtained and the relative percentage of HBc⁺ cells was calculated by normalizing to the virus-only control well in the same lane. For example, the absolute HBc⁺ cell percentage in virus-only control well (considered as reference) was 40%, while adding one neutralizing serum sample might reduce this to 10%. Therefore, after normalization, the relative percentages of HBc⁺ cells were calculated as 100% in control well and 25% for this neutralizing serum sample. Since many factors (virus concentration, cell concentration, immunofluorescence reading, etc.) vary between different plates or different rounds of experiments, normalization is necessary for combining data for comparison.

ELISA Assays

Blood samples were submitted to Memorial Sloan Kettering Cancer Center for clinical testing. The presence of HBsAg protein and anti-HBc antibody, as well as anti-HBs titers, were determined by ELISA (Abbott Laboratories) as per the manufacturer's instructions.

The binding of serum or recombinant IgG antibodies to HBsAg proteins (see KEY RESOURCES TABLE) was measured by coating ELISA plates with 10 μg/ml of antigen in PBS. Plates were blocked with 2% BSA in PBS and incubated with antibody for one hour at room temperature. Visualization was with HRP-conjugated goat anti-human IgG (Thermo Fisher Scientific). The 50% effective concentration (EC₅₀) needed for maximal binding was determined by non-linear regression analysis in software PRISM.

For competition ELISAs plates were coated with 0.12 μg/m1HBsAg (adr CHO) and incubated with 16.7 μg/ml primary antibody for two hours, followed by directly adding 0.25 μg/ml biotinylated secondary antibody and incubation for 30 minutes all at room temperature. Detection was with streptavidin-HRP (BD Biosciences).

Autoreactivity and Polyreactivity

Autoreactivity and polyreactivity assays were performed as described (Gitlin et al., 2016; Mayer et al., 2017; Robbiani et al., 2017). For the autoreactivity assays, monoclonal antibodies were tested with the Antinuclear antibodies (HEp-2) Kit (MBL International). Antibodies were incubated at 100 μg/ml and were detected with Alexa Fluor 488 AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch) at 10 μg/ml. Fluorescence images were taken with a wide-field fluorescence microscope (Axioplan 2, Zeiss), a 40× dry objective and a Hamamatsu Orca ER B/W digital camera. Images were analyzed with Image J. Human serum containing antinuclear antibodies (MBL International) was used as a positive control. For the polyreactivity ELISA assays, antibody binding to five different antigens, double-stranded DNA (dsDNA), insulin, keyhole limpet hemocyanin (KLH), lipopolysaccharides (LPS), and single-stranded DNA (ssDNA), were measured. ED38 (Wardemann et al., 2003) and mG053 (Yurasov et al., 2005) antibodies were used as positive and negative controls, respectively.

Synthetic Peptides

Eighteen peptides spanning the antigenic loop region of S-protein antigen were synthesized at the Proteomics Resource Center of The Rockefeller University. For peptide ELISAs plates were coated with 10 μg/ml peptide in PBS.

HBsAg-Binding Memory B Cells

S-protein (adr serotype) expressed and purified from Chinese hamster ovary (CHO) cells (ProSpec) and ovalbumin (Sigma-Aldrich) were biotinylated using EZ-Link™ Micro NHS-PEG4-Biotinylation kit (Thermo Fisher Scientific). S-protein-PE and S-protein-APC were prepared by incubating 2-3 μg of biotin-S-protein with streptavidin-PE (eBioscience) or streptavidin-APC (BD Biosciences) in PBS respectively overnight at 4° C. in the dark. Ovalbumin-Alexa Fluor 488 was generated by incubating biotin-ovalbumin with streptavidin-Alexa Fluor 488 (Thermo Fisher Scientific).

B cell purification, labeling, and sorting were as previously described (Escolano et al., 2019; Robbiani et al., 2017; Tiller et al., 2008; von Boehmer et al., 2016). Briefly, PBMCs were thawed and washed with RPMI medium at 37° C. B lymphocytes were positively selected using CD19 MicroBeads (Miltenyi Biotec) followed by incubation with human Fc block (BD Biosciences) and anti-CD20-PECy7 (BD Biosciences), anti-IgG-Bv421 (BD Biosciences), S-protein-PE at 10 μg/ml, S-protein-APC at 10 μg/ml, and ovalbumin-Alexa Fluor 488 at 10 μg/ml at 4° C. for 20 minutes. Single CD20⁺ IgG⁺ S-protein-PE⁺ S-protein-APC⁺ Ova-Alexa Fluor 488⁻ memory B cells were sorted into 96-well plates using a FACSAriaII (Becton Dickinson) and stored at −80° C.

Antibody Cloning, Sequencing and Production

Antibody cloning, sequencing and production were done as previously reported (Robbiani et al., 2017; Tiller et al., 2008; von Boehmer et al., 2016). Primers are listed in Table S3. Unmutated common ancestor (UCA) antibody sequences of H006, H019 and H020 were synthesized by gBlock IDT (Table S3) and were inserted into antibody vectors for expression. V(D)J gene segment and CDR3 sequences were determined by IgBlast (Ye et al., 2013) and/or IMGT/V-QUEST (Brochet et al., 2008).

S-Protein Mutagenesis

Oligonucleotides fragments with the target point mutations were synthesized by gBlock IDT (Table S3), and were substituted into the antigenic loop region in plasmid p1.3×HBV-WT by Sequence and Ligation-Independent Cloning (SLIC) (Jeong et al., 2012). Mutant plasmids were transfected into Huh-7.5-NTCP cells using X-tremeGENE 9 DNA Transfection Reagent (Sigma-Aldrich) and the culture medium was changed to serum-free DMEM after 24 hours. Supernatants were collected 2 days later and stored at −80° C. Serum-free medium (50 μl) was directly used to coat ELISA plates.

Crystallization, X-Ray Data Collection, Structure Determination and Refinement

Antibody Fab (25 mg/ml) in 50 mM Tris 8.0, 50 mM NaCl was mixed with peptide (5 mg/ml) in the same buffer at 5:1 v/v. Molar ratio of Fab:peptide is around 1:2. Crystals were obtained upon substitution of all peptide-11 cysteine residues with serine in the peptide synthesis (Proteomics Resource Center, RU). The crystallization condition for Fab15/peptide-11Ser was identified from a commercial screen (Morpheus by Molecular Dimensions) by the sitting-drop vapor-diffusion method at room temperature. The crystal used for data collection was obtained directly from the initial setup (position E1) in a precipitant solution consisting of 0.12 M Ethylene glycols (Di, Tri, Tetra and Penta-ethylene glycol), 0.1 M Buffer Mix 1 (Imidazole/MES) at pH 6.5 and 30% Precipitant Mix 1 (20% v/v PEG 500* MME; 10% w/v PEG 20000). The crystals were flash-cooled in liquid nitrogen directly from the mother liquor without additional cryoprotectant. X-ray diffraction data were collected from a single crystal on the Advanced Photon Source (APS) beamline 24-ID-E to 1.78 Å resolution. The data were integrated and scaled with the program XDS (Kabsch, 2010a, b) and other data processing utilities from the CCP4 suite (Collaborative Computational Project, 1994) using RAPD, the software available at the beam-line. Initial phase estimates and electron-density maps were obtained by molecular replacement with Phaser (McCoy et al., 2007) using a single FAB molecule from (PDB: SGGU) as an initial search model in Phenix (Adams et al., 2010). Iterative model building and structural refinement were manually performed using COOT (Emsley et al., 2010) and Phenix, respectively. The peptide density was well defined, and refined to 90% occupancy, for residues STKPSDGNST (SEQ ID NO: 25). All other residues were not visible and the area where they would be is fully solvent, with no crystal contacts involving any of the peptide atoms. The quality of the final model was good as noted in a Ramachandran of 96% of the observed residues within the allowable region. Data-collection and refinement statistics are summarized (FIG. 13B). All molecular graphics were prepared with PyMOL (Version 2.0 Schrödinger, LLC). Atomic coordinates and experimental structure factors have been deposited in the PDB under accession code 6VJT.

Humanized Mice and In Vivo Studies

Six to eight week old Fah^(−/−) NODRag1^(−/−) IL2rg^(null) (FNRG) female mice were transplanted with one million human hepatocytes from a pediatric female donor HUM4188 (Lonza Bioscience) as previously described (de Jong et al., 2014). Briefly, during isoflurane anesthesia mice underwent skin and peritoneal incision, exposing the spleen. One million hepatocytes were injected in the spleen using a 28-gauge needle. The peritoneum was then approximated using 4.0 VICRYL sutures (Johnson & Johnson), and skin was closed using MikRon Autoclip surgical clips (Becton Dickinson). Mice were cycled off the drug nitisinone (Yecuris) on the basis of weight loss and overall health. Humanization was monitored by human albumin quantification in mouse serum using a human-specific ELISA (Bethyl Labs). Humanized FNRG mice with human albumin values greater than 1 mg/ml were used for infection experiments. The human liver chimeric (huFNRG) mice are extremely immunodeficient. The Rag1^(−/−) renders the mice B and T cell deficient and the IL2rg^(null) mutation prevents cytokine signaling through multiple receptors, leading to a deficiency in functional NK cells. Moreover, the genetic background is NOD background, with suboptimal antigen presentation, defects in T and NK cell function, reduced macrophage cytokine production, suppressed wound healing, and C5 complement deficiency. Thus the mice would be unable to produce antibody-dependent effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), or passive antibody-enhanced adaptive immunity.

Mice were challenged intravenously with 1×10⁴ genome equivalent (GE) of mouse-passaged genotype C HBV viruses diluted in PBS. For prophylaxis experiments, 500 μg of monoclonal antibody was administered intraperitoneally at 20 and again at 6 hours before infection. For therapy experiments, huFNRG mice with established HBV infections (<10⁸ DNA copies/ml of serum) were injected with 500 μg of each monoclonal antibody intraperitoneally 3 times per week.

DNA in mouse serum collected weekly was extracted using a QIAamp DNA Blood Mini Kit (Qiagen). Total HBV DNA was determined by quantitative PCR (Michailidis et al., 2017). PCR was performed using a TaqMan Universal PCR Master Mix (Applied Biosystems), primers and probe (Table S3).

To obtain HBV DNA from serum for sequence analysis the S domain was amplified using primers (Table S3), and Phusion DNA polymerase (Thermo Fisher Scientific). Initial denaturation was at 98° C. for 30 s, followed by 40 amplification cycles (98° C. for 10 s, 60° C. for 30 s, and 72° C. for 30 s), followed by one cycle at 72° C. for 5 min. A ˜700 bp fragment was gel extracted for Sanger sequencing. Sequence alignments were performed using MacVector.

Quantification and Statistical Analysis

The detailed information of statistical analysis could be found in the Result and Figure Legends. Correlation was evaluated by Spearman's rank correlation method (FIGS. 1A and 2B). Statistical significance was calculated by Dunn's Kruskal-Wallis multiple comparisons with p values corrected with the Benjamini-Hochberg procedure (FIG. 8C). The 50% effective concentration (EC₅₀) values by ELISA assays (FIGS. 3A and 3C) and 50% inhibitory concentration (IC₅₀) values by neutralization assays (FIG. 5C) were calculated by nonlinear regression analysis in PRISM software.

DISCUSSION OF EXAMPLES

Previous studies have identified several anti-HBs neutralizing antibodies from a small number of otherwise unselected spontaneously recovered or vaccinated individuals (Cerino et al., 2015; Colucci et al., 1986; Eren et al., 1998; Heijtink et al., 2002; Heijtink et al., 1995; Jin et al., 2009; Kim and Park, 2002; Li et al., 2017; Sa'adu et al., 1992; Sankhyan et al., 2016; Tajiri et al., 2007; Tokimitsu et al., 2007; Wang et al., 2016). In contrast, in the present disclosure, sera from 144 exposed volunteers was screened to identify elite neutralizers. Serologic activity varied greatly among the donors with a small number of individuals demonstrating high levels of neutralizing activity. To understand this activity, we isolated 244 anti-HBs antibodies from single B cells obtained from the top donors. Each of the elite donors tested showed expanded clones of memory B cells expressing bNAbs that targeted 3 non-overlapping sites on the S-protein. Moreover, the amino acid sequence of several of the bNAbs was highly similar in different individuals. These closely related antibodies target the same epitope.

The near identity of clones of HBV bNAbs in unrelated elite individuals is akin to reports for elite responders to HIV-1 (Scheid et al., 2011; West et al., 2012), influenza (Laursen and Wilson, 2013; Pappas et al., 2014; Wrammert et al., 2011), Zika (Robbiani et al., 2017), and malaria (Tan et al., 2018). However, none of the elite anti-HBs bNAbs shares both IgH and IgL with previously reported HBV neutralizing antibodies, the best of which have been tested in the clinic but are less potent than some of the bNAbs of this disclosure (libivirumab IC₅₀: 35 ng/ml, tuvirumab IC₅₀: ˜100 ng/ml) (Galun et al., 2002; Heijtink et al., 2001; van Nunen et al., 2001).

The described alanine scanning and competition binding analyses are consistent with the existence of at least 3 domains that can be recognized concomitantly by bNAbs (Gao et al., 2017; Tajiri et al., 2010; Zhang et al., 2016). However, the domains do not appear to be limited to either of two previously defined circular peptide epitopes, 123-137 and 139-148 (Tajiri et al., 2010; Zhang et al., 2016). Instead, residues spanning most of the external domain can contribute to binding by both Group-I and -II antibodies. For example, alanine scanning indicates that Group-I H020 binding is dependent on I110, K141, D144, G145 and T148, while Group-II H016 binding depends on T123, D144, and G145. Thus, despite having non-overlapping binding sites some of the essential residues are shared by Group-I and II suggesting that the epitopes are conformational. Moreover, the antibody epitopes on S-protein identified using mouse and human antibodies may be distinct (Chen et al., 1996; Ijaz et al., 2003; Paulij et al., 1999; Zhang et al., 2019; Zhang et al., 2016). Finally, G145, a residue that is frequently mutated in infected humans (Ma and Wang, 2012; Tong et al., 2013), is believed to be essential for binding by all the Group-II but not all Group-I or -III antibodies tested.

Crystallization of the Group-II bNAb H015 and its linear epitope revealed a loop that includes P142, S/T143, D144, and G145, all of which are frequently mutated during natural infection to produce well-documented immune escape variants (Hsu et al., 2015; Ijaz et al., 2012; Ma and Wang, 2012; Salpini et al., 2015). In addition to immune escape, the residues that form this structure are also essential for infectivity, possibly by facilitating virus interactions with cell surface glycosaminoglycans (Sureau and Salisse, 2013). Mutations in K141, P142 as well as C139 and C147, all of which contribute to the stability of the structure, decrease viral infectivity (Salisse and Sureau, 2009). Without intending to be bound by any particular theory, it is considered that drugs that destabilize the newly elucidated H015-peptide loop structure may also interfere with infectivity.

The G145R mutation, which is among the most frequent immune escape variants, replaces a small neutral residue with a bulky charged residue that would likely interfere with antigenicity by destroying the salt bridge between K141 and D144 that anchors the peptide loop. However, this drastic structural change does not alter infectivity (Salisse and Sureau, 2009), possibly because the additional charge compensates for otherwise altered interactions between HBV and cell surface glycosaminoglycans (Sureau and Salisse, 2013). Thus, the additional charge may allow G145R to function as a dominant immune escape variant while preserving infectivity.

The present disclosure describes antibodies directed at S-protein antigen in part because this is the antigen used in the currently FDA-approved vaccines, and because purified S-protein blocked nearly all of the neutralizing activity in the serum of the elite neutralizers irrespective of whether they were vaccinated or spontaneously recovered. Nevertheless, individuals who recover from infection also produce antibodies to the PreS1 domain of HBsAg (Li et al., 2017; Sankhyan et al., 2016). The PreS1 domain is essential for the virus to interact with the entry factor NCTP on hepatocytes and potent neutralizing antibodies to PreS1 have been described (Li et al., 2017). However, these are not naturally occurring antibodies but rather randomly paired IgH and IgL chains derived from phage libraries obtained from unexposed or vaccinated healthy donors (Li et al., 2017). Moreover, the phage antibodies required further engineering to enhance their neutralizing activity (Li et al., 2017). Thus, whether the human immune system also produces potent anti-PreS1 bNAbs has not been determined.

Chronic HBV infection remains a major global public health problem in need of an effective curative strategy (Graber-Stiehl, 2018; Lazarus et al., 2018; Revill et al., 2016). Chronically infected individuals produce an overwhelming amount of HBsAg that is postulated to incapacitate the immune system. Consequently, immune cells, which might normally clear the virus, are unable to react to antigen, a phenomenon referred to as exhaustion or anergy (Ye et al., 2015). The appearance of anti-HBs antibodies is associated with spontaneous recovery from the disease, perhaps because they can clear the antigen and facilitate the emergence of a productive immune response (Celis and Chang, 1984; Zhang et al., 2016; Zhu et al., 2016). These findings led to the hypothesis that passively administered antibodies might be used in conjunction with antiviral drugs to further decrease the antigenic burden while enhancing immune responses that maintain long-term control of the disease. The presently described results in huFNRG mice infected with HBV indicate that antibody monotherapy with a potent bNAb can lead to the emergence of the very same escape mutations commonly found in chronically infected individuals. Moreover, not all bNAb combinations are effective in preventing escape by mutation. Combinations that target separate epitopes but have overlapping sensitivity to commonly occurring escape mutations such as H006 and H007 are ineffective. In contrast, combinations with complementary sensitivity to common escape mutations prevent the emergence of escape mutations in huFNRG mice infected with HBV. Thus, as described above, the present disclosure provides immunotherapy for HBV infection with combinations of antibodies with complementary activity to avert this potential problem.

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TABLE 51 Detailed information of donors, Related to Figure 1. The anti-HBs ELISA titer (x-axis in Figure 1A and 8B) and relative infection rate (y-axis in Figure 1A and 8B) of each donor's serum sample were determined by ELISA assay and in vitro neutralization assay, respectively. ANTI- HBs INFECTION RATE INFECTION RATE ELISA BIG (1:5 SERUM) NEUTRA (1:50 SERUM) NEUTRA DONOR TITE STAT- BLOOD PERCENTAGE AVER- CAPAC- PERCENTAGE AVER- CAPAC- ID R UE DRAW OF HBcAg + CELLS AGE ITY OF HBcAg + CELLS AGE ITY QWA- 9.08 Vacci- 114.00 127.12 133.64 124.92 0.80 118.06 128.90 122.19 123.05 0.81 0947- nated 001 QWA- 516.12 Vacci- 65.28 77.58 75.13 72.66 1.38 131.60 137.04 133.44 134.03 0.75 0947- nated 002 QWA- 0 Vacci- 108.59 118.31 118.53 115.14 0.87 132.81 136.65 132.22 133.89 0.75 0947- nated 003 QWA- 267.1 Vacci- 113.39 121.91 121.67 118.99 0.84 142.69 146.69 153.24 147.54 0.68 0947- nated 004 QWA- 113.01 Vacci- 82.91 87.48 95.39 88.59 1.13 148.24 149.36 146.75 148.12 0.68 0947- nated 005 QWA- 325.77 Vacci- 81.19 85.97 96.43 87.86 1.14 117.48 116.83 119.74 118.02 0.85 0947- nated 006 QWA- 63.54 Vacci- 100.37 105.53 110.73 105.54 0.95 134.11 135.15 138.51 135.92 0.74 0947- nated 007 QWA- 21.8 Vacci- 40.86 35.60 42.82 39.76 2.52 78.64 84.09 84.79 82.51 1.21 0947- nated 008 QWA- >1000 Vacci- BIG 12.84 14.39 17.20 14.81 6.75 34.66 38.81 33.48 35.65 2.81 0947- nated BLOOD 009 DRAW QWA- 0.33 Vacci- 93.33 93.42 104.91 97.22 1.03 105.41 116.72 115.58 112.57 0.89 0947- nated 010 QWA- 1.29 Vacci- BIG 109.40 113.87 94.10 105.79 0.95 95.79 93.25 81.95 90.33 1.11 0947- nated BLOOD 011 DRAW QWA- 12.87 Vacci- 102.04 102.94 108.81 104.60 0.96 142.95 137.95 125.12 135.34 0.74 0947- nated 012 QWA- >1000 Vacci- BIG 9.24 8.31 8.33 8.63 11.59 19.95 20.81 17.29 19.35 5.17 0947- nated BLOOD 013 DRAW QWA- 248.31 Vacci- 80.90 80.65 79.88 80.48 1.24 117.58 120.17 115.37 117.71 0.85 0947- nated 014 QWA- 76.1 Vacci- 97.26 91.48 86.95 91.90 1.09 112.79 109.82 101.19 107.93 0.93 0947- nated 015 QWA- 0.36 Non- 100.22 96.77 95.84 97.61 1.02 118.04 120.05 117.34 118.48 0.84 0947- Vacci- 016 nated QWA- 0.63 Vacci- 100.30 102.98 101.55 101.61 0.98 122.27 119.42 110.93 117.54 0.85 0947- nated 017 QWA- 3.24 Vacci- 101.65 113.38 96.98 104.00 0.96 118.18 121.26 107.05 115.50 0.87 0947- nated 018 QWA- >1000 Vacci- 47.62 43.35 40.11 43.69 2.29 105.86 100.46 89.54 98.62 1.01 0947- nated 019 QWA- 1.21 Non- 77.13 75.25 60.04 70.81 1.41 116.28 113.72 108.65 112.88 0.89 0947- Vacci- 020 nated QWA- 15.81 Vacci- 33.85 41.39 34.98 36.74 2.72 89.74 89.87 88.60 89.40 1.12 0947- nated 021 QWA- 2.17 Vacci- 83.52 94.22 93.88 90.54 1.10 100.08 119.88 110.19 110.05 0.91 0947- nated 022 QWA- 183.37 Vacci- 93.15 119.57 116.08 109.60 0.91 113.43 117.91 103.45 111.60 0.90 0947- nated 023 QWA- 256.85 Vacci- 55.70 60.03 49.13 54.95 1.82 104.68 116.21 105.17 108.69 0.92 0947- nated 024 QWA- 709.29 Vacci- 76.18 77.38 75.01 76.19 1.31 100.58 105.57 116.82 107.66 0.93 0947- nated 025 QWA- 0.49 Non- 83.02 102.42 96.98 94.14 1.06 104.24 117.09 105.73 109.02 0.92 0947- Vacci- 026 nated QWA- 0.3 Non- BIG 103.51 128.79 114.82 115.71 0.86 105.91 126.68 117.03 116.54 0.86 0947- Vacci- BLOOD 027 nated DRAW QWA- 6.51 Non- 84.03 90.88 87.77 87.56 1.14 107.23 117.89 108.21 111.11 0.90 0947- Vacci- 028 nated QWA- 1.67 Non- 103.72 98.68 83.74 95.38 1.05 96.06 102.25 100.20 99.50 1.00 0947- Vacci- 029 nated QWA- 0.09 Vacci- BIG 86.06 84.44 91.73 87.41 1.14 111.74 115.73 102.26 109.91 0.91 0947- nated BLOOD 030 DRAW QWA- 488.3 Core 71.30 66.21 56.71 64.74 1.54 94.99 102.87 94.27 97.38 1.03 0947- Ab + 031 QWA- >1000 Vacci- 71.12 67.87 56.53 65.17 1.53 97.45 107.81 106.59 103.95 0.96 0947- nated 032 QWA- 330.93 Vacci- 99.61 93.60 87.25 93.49 1.07 106.28 116.65 108.45 110.46 0.91 0947- nated 033 QWA- 588.26 Core 40.92 41.19 35.30 39.14 2.56 99.54 111.16 105.28 105.32 0.95 0947- Ab + 034 QWA- 365.7 Vacci- 101.52 113.83 94.76 103.37 0.97 129.77 131.60 121.19 127.52 0.78 0947- nated 035 QWA- 108.83 Vacci- 87.55 85.69 77.65 83.63 1.20 122.28 122.03 107.69 117.33 0.85 0947- nated 036 QWA- >1000 Vacci- 23.79 21.09 18.46 21.11 4.74 66.02 59.97 52.64 59.54 1.68 0947- nated 037 QWA- 11.76 Vacci- 94.89 95.53 96.87 95.76 1.04 119.79 125.09 115.27 120.05 0.83 0947- nated 038 QWA- 230.99 Vacci- 102.08 99.23 91.54 97.62 1.02 113.01 115.04 98.95 109.00 0.92 0947- nated 039 QWA- 0.26 Vacci- 54.12 57.38 45.32 52.27 1.91 102.43 107.89 95.37 101.90 0.98 0947- nated 040 QWA- 0.42 Non- 77.09 78.29 84.91 80.09 1.25 87.59 101.25 101.19 96.68 1.03 0947- Vaccin 041 ated QWA- 128.75 Core 97.03 106.51 110.26 104.60 0.96 114.07 149.01 121.98 128.35 0.78 0947- Ab + 042 QWA- 229.75 Core 78.56 94.35 95.71 89.54 1.12 95.18 144.23 123.82 121.08 0.83 0947- Ab + 043 QWA- 222.48 Core 45.85 56.61 65.34 55.93 1.79 94.00 133.96 109.86 112.60 0.89 0947- Ab + 044 QWA- >1000 Core 51.82 66.02 61.55 59.80 1.67 104.36 122.03 120.96 115.78 0.86 0947- Ab + 045 QWA- 350.93 Vacci- 89.57 99.40 111.55 100.17 1.00 100.98 129.50 111.79 114.09 0.88 0947- nated 046 QWA- 0.33 Vacci- 126.88 138.30 142.37 135.85 0.74 112.35 156.77 127.72 132.28 0.76 0947- nated 047 QWA- >1000 Vacci- BIG 35.94 44.44 46.02 42.14 2.37 83.08 109.89 94.60 95.86 1.04 0947- nated BLOOD 048 DRAW QWA- >1000 Core BIG 94.21 120.65 119.57 111.48 0.90 113.33 139.10 126.43 126.28 0.79 0947- Ab + BLOOD 049 DRAW QWA- 6.2 Vacci- 93.10 114.26 113.58 106.98 0.93 93.34 121.07 112.91 109.10 0.92 0947- nated 050 QWA- 228.35 Vacci- 111.78 112.69 115.97 113.48 0.88 104.71 102.64 83.60 96.98 1.03 0947- nated 051 QWA- 2.77 Vacci- 116.53 115.59 110.83 114.32 0.87 113.48 114.25 125.60 117.78 0.85 0947- nated 052 QWA- 48.25 Vacci- 114.90 112.78 98.40 108.69 0.92 105.04 116.61 119.01 113.55 0.88 0947- nated 053 QWA- 24.06 Vacci- 121.56 125.13 111.71 119.47 0.84 99.64 118.99 122.11 113.58 0.88 0947- nated 054 QWA- 772.43 Core BIG 8.79 9.33 5.78 7.97 12.55 82.33 95.93 87.80 88.69 1.13 0947- Ab + BLOOD 055 DRAW QWA- 441.12 Vacci- 86.57 86.67 79.10 84.12 1.19 98.81 120.31 116.13 111.75 0.89 0947- nated 056 QWA- 91.37 Vacci- 120.46 113.18 110.80 114.82 0.87 111.45 107.08 116.80 111.78 0.89 0947- nated 057 QWA- >1000 Vacci- 27.66 27.74 26.37 27.26 3.67 82.86 83.73 74.74 80.44 1.24 0947- nated 058 QWA- 83.28 Vacci- 110.69 109.95 99.34 106.66 0.94 115.21 115.69 99.96 110.29 0.91 0947- nated 059 QWA- >1000 Vacci- BIG 5.68 6.72 5.17 5.86 17.08 25.39 26.65 24.93 25.66 3.90 0947- nated BLOOD 060 DRAW QWA- 104.78 Vacci- 109.98 120.25 131.62 120.61 0.83 107.59 134.01 124.63 122.08 0.82 0947- nated 061 QWA- 2.56 Vacci- 113.16 140.19 143.78 132.38 0.76 120.58 139.98 135.32 131.96 0.76 0947- nated 062 QWA- 246.16 Vacci- 101.93 112.01 141.39 118.44 0.84 114.71 134.13 143.18 130.67 0.77 0947- nated 063 QWA- Not Vacci- 128.17 114.29 147.09 129.85 0.77 133.50 133.31 133.23 133.35 0.75 0947- Avail- nated 064 able QWA- 0.28 Core 112.39 133.12 155.43 133.65 0.75 131.76 134.78 150.74 139.09 0.72 0947- Ab + 065 QWA- 265.6 Vacci- 50.87 68.37 86.59 68.61 1.46 129.24 133.43 119.16 127.28 0.79 0947- nated 066 QWA- 0 Vacci- 106.36 120.57 150.56 125.83 0.79 122.57 142.42 139.51 134.84 0.74 0947- nated 067 QWA- 0.25 Vacci- 58.26 87.26 105.25 83.59 1.20 123.65 128.77 124.58 125.67 0.80 0947- nated 068 QWA- >1000 Vacci- BIG 11.42 27.20 22.32 20.31 4.92 50.80 55.44 64.27 56.84 1.76 0947- nated BLOOD 069 DRAW QWA- 7.01 Vacci- 107.39 113.96 138.16 119.84 0.83 118.06 116.97 118.95 117.99 0.85 0947- nated 070 QWA- 638.24 Vacci- 56.39 48.55 55.42 53.45 1.87 94.83 111.19 117.23 107.75 0.93 0947- nated 071 QWA- 15.55 Vacci- 128.74 135.08 126.34 130.05 0.77 137.00 132.93 122.94 130.96 0.76 0947- nated 072 QWA- 225.34 Vacci- 131.87 122.33 137.50 130.57 0.77 137.43 128.35 124.59 130.12 0.77 0947- nated 073 QWA- 159.52 Core 120.51 119.04 128.75 122.76 0.81 140.98 130.18 124.00 131.72 0.76 0947- Ab + 074 QWA- 0.37 Vacci- 131.78 125.05 159.51 138.78 0.72 133.08 135.29 117.74 128.70 0.78 0947- nated 075 QWA- 27.37 Vacci- 137.32 145.89 141.60 0.71 132.06 132.06 0.76 0947- nated 076 QWA- 52.77 Vacci- 112.72 121.48 117.10 0.85 137.12 137.12 0.73 0947- nated 077 QWA- 181.6 Vacci- 116.16 107.29 111.72 0.90 138.30 131.27 134.79 0.74 0947- nated 078 QWA- >1000 Vacci- 54.73 59.28 71.14 61.72 1.62 130.53 128.66 118.32 125.84 0.79 0947- nated 079 QWA- >1000 Vacci- 62.38 53.55 69.34 61.76 1.62 122.37 106.42 114.40 0.87 0947- nated 080 QWA- 7.23 Vacci- 113.81 111.55 142.59 122.65 0.82 109.38 105.63 116.78 110.60 0.90 0947- nated 081 QWA- 24.98 Vacci- 120.69 124.58 118.10 121.12 0.83 106.45 118.55 117.79 114.26 0.88 0947- nated 082 QWA- 90.5 Vacci- 84.79 87.62 99.65 90.69 1.10 91.86 99.93 105.67 99.15 1.01 0947- nated 083 QWA- 880.92 Core 112.81 125.10 114.29 117.40 0.85 106.78 7.78 147.46 124.01 0.81 0947- Ab + 084 QWA- 82.25 Vacci- 120.45 148.83 159.96 143.08 0.70 114.14 114.33 136.45 121.64 0.82 0947- nated 085 QWA- 19.16 Vacci- 147.29 173.59 166.75 162.55 0.62 110.03 128.28 120.46 119.59 0.84 0947- nated 086 QWA- 88.6 Vacci- 126.58 146.46 145.74 139.59 0.72 105.86 119.12 119.22 114.74 0.87 0947- nated 087 QWA- 288.42 Core 108.25 120.39 140.09 122.91 0.81 90.55 101.28 115.15 102.33 0.98 0947- Ab + 088 QWA- 15.74 Vacci- 101.12 94.87 98.43 98.14 1.02 75.70 96.65 111.21 94.52 1.06 0947- nated 089 QWA- >1000 Vacci- 54.59 47.73 53.63 51.99 1.92 71.09 87.31 99.22 85.87 1.16 0947- nated 090 QWA- >1000 Vacci- 18.28 23.05 19.85 20.39 4.90 48.17 59.50 53.15 53.61 1.87 0947- nated 091 QWA- 2.64 Vacci- 155.82 147.87 143.25 148.98 0.67 126.83 113.67 124.04 121.51 0.82 0947- nated 093 QWA- 127.58 Vacci- 160.76 145.63 119.61 142.00 0.70 105.84 110.91 114.14 110.30 0.91 0947- nated 094 QWA- 23.74 Vacci- 176.74 160.65 159.27 165.55 0.60 110.83 103.36 112.17 108.78 0.92 0947- nated 095 QWA- >1000 Vacci- 51.24 50.60 36.36 46.07 2.17 109.95 99.89 100.76 103.54 0.97 0947- nated 096 QWA- 25.97 Vacci- 151.86 144.37 127.26 141.16 0.71 109.63 109.86 98.50 106.00 0.94 0947- nated 097 QWA- >1000 Vacci- 73.82 83.70 70.82 76.11 1.31 88.55 95.98 100.28 94.94 1.05 0947- nated 098 QWA- 688.28 Vacci- BIG 110.48 112.47 90.05 104.33 0.96 100.29 103.59 94.65 99.51 1.00 0947- nated BLOOD 099 DRAW QWA- 366.05 Vacci- 85.68 99.38 86.23 90.43 1.11 97.85 101.09 85.62 94.85 1.05 0947- nated 100 QWA- 1.22 Vacci- 88.54 96.48 92.77 92.60 1.08 107.51 104.47 106.69 106.22 0.94 0947- nated 101 QWA- 0.65 Vacci- 96.90 94.97 99.88 97.25 1.03 109.63 101.73 102.43 104.60 0.96 0947- nated 102 QWA- 45.53 Core 100.38 104.13 93.37 99.29 1.01 103.40 95.77 93.83 97.67 1.02 0947- Ab + 103 QWA- 95.76 Vacci- 95.99 87.67 79.38 87.68 1.14 111.24 105.95 113.01 110.07 0.91 0947- nated 104 QWA- 1.48 Vacci- 42.47 43.05 47.61 44.38 2.25 102.15 99.45 98.24 99.95 1.00 0947- nated 105 QWA- >1000 Vacci- 16.40 16.66 16.15 16.40 6.10 64.77 68.47 66.59 66.61 1.50 0947- nated 106 QWA- 32.84 Vacci- 148.16 141.65 127.35 139.05 0.72 123.79 119.06 104.81 115.89 0.86 0947- nated 107 QWA- 352.99 Vacci- 78.23 81.50 84.50 81.41 1.23 109.31 116.46 103.38 109.72 0.91 0947- nated 108 QWA- 15.04 Vacci- 112.45 103.15 97.70 104.43 0.96 110.85 121.08 117.28 116.40 0.86 0947- nated 109 QWA- 26.27 Vacci- 86.31 102.24 99.68 96.08 1.04 115.57 130.13 116.61 120.77 0.83 0947- nated 110 QWA- 40.79 Vacci- 81.44 86.98 87.80 85.40 1.17 109.93 98.54 91.77 100.08 1.00 0947- nated 111 QWA- 260.82 Core 35.62 36.63 32.67 34.97 2.86 69.44 65.66 65.06 66.72 1.50 0947- Ab + 112 QWA- 1.97 Vacci- 93.25 98.77 81.76 91.26 1.10 101.75 104.27 100.12 102.05 0.98 0947- nated 113 QWA- >1000 Vacci- 32.49 27.64 25.33 28.49 3.51 80.93 61.56 71.59 71.36 1.40 0947- nated 114 QWA- 798.12 Core 13.86 14.67 15.02 14.52 6.89 45.62 46.82 42.16 44.87 2.23 0947- Ab + 115 QWA- 459.84 Vacci- 99.96 93.67 81.32 91.65 1.09 107.55 108.08 109.31 108.31 0.92 0947- nated 116 QWA- 0.46 Vacci- 98.06 92.72 95.42 95.40 1.05 106.12 104.66 111.74 107.51 0.93 0947- nated 117 QWA- 20.87 Vacci- 151.82 146.04 144.65 147.50 0.68 112.36 109.15 124.29 115.26 0.87 0947- nated 118 QWA- 91.47 Vacci- 114.36 109.63 108.87 110.96 0.90 108.91 112.79 104.89 108.86 0.92 0947- nated 119 QWA- 13 Core 44.97 45.06 40.30 43.44 2.30 102.35 110.61 97.70 103.55 0.97 0947- Ab + 120 QWA- 175.21 Vacci- 91.44 91.91 96.95 93.43 1.07 166.31 164.27 144.97 158.52 0.63 0947- nated 121 QWA- 51.51 Vacci- 95.47 92.45 100.97 96.30 1.04 180.79 174.92 143.73 166.48 0.60 0947- nated 122 QWA- 36.81 Vacci- 145.77 156.75 146.88 149.80 0.67 180.84 176.71 135.18 164.24 0.61 0947- nated 123 QWA- 1.3 Vacci- 147.11 133.31 131.87 137.43 0.73 170.89 169.84 127.57 156.10 0.64 0947- nated 124 QWA- 1.44 Vacci- 110.99 111.85 118.19 113.68 0.88 178.09 178.48 149.45 168.67 0.59 0947- nated 125 QWA- 359.06 Core 79.16 76.31 85.67 80.83 1.24 167.98 161.85 154.57 161.47 0.62 0947- Ab + 126 QWA- 1.23 Non- 128.75 125.40 143.54 132.56 0.75 162.01 157.22 139.08 152.77 0.65 0947- Vacci- 127 nated QWA- >1000 Vacci- 30.63 25.86 36.31 30.94 3.23 69.73 82.09 84.44 78.76 1.27 0947- nated 128 QWA- 0.17 Non- 166.56 156.86 157.89 160.44 0.62 162.24 168.18 154.52 161.65 0.62 0947- Vacci- 129 nated QWA- 11.81 Vacci- 143.14 129.67 114.98 129.26 0.77 156.94 144.37 119.26 140.19 0.71 0947- nated 131 QWA- 0 Non- 143.37 148.51 134.41 142.09 0.70 170.72 146.03 126.56 147.77 0.68 0947- Vacci- 132 nated QWA- 731.27 Core 86.48 85.37 78.16 83.34 1.20 157.64 137.87 131.07 142.20 0.70 0947- Ab + 133 QWA- >1000 Vacci- 31.62 30.52 27.60 29.92 3.34 144.36 137.82 115.14 132.44 0.76 0947- nated 134 QWA- 796.93 Core 81.56 81.84 88.25 83.88 1.19 157.58 142.42 116.84 138.95 0.72 0947- Ab + 135 QWA- 0 Non- 128.19 132.95 139.91 133.68 0.75 165.85 140.56 137.58 148.00 0.68 0947- Vacci- 136 nated QWA- 1.56 Non- 155.09 151.93 137.05 148.02 0.68 175.51 155.22 128.75 153.16 0.65 0947- Vacci- 137 nated QWA- 0.74 Non- 160.76 159.87 135.61 152.08 0.66 165.30 137.96 119.52 140.93 0.71 0947- Vacci- 138 nated QWA- 0 Non- 143.96 145.71 123.98 137.88 0.73 143.82 125.60 114.22 127.88 0.78 0947- Vacci- 139 nated QWA- 609.46 Core 93.61 96.21 89.99 93.27 1.07 125.42 135.92 106.58 122.64 0.82 0947- Ab + 140 QWA- >1000 Vacci- 35.14 37.74 36.31 36.40 2.75 90.32 87.71 83.80 87.28 1.15 0947- nated 141 QWA- 0.01 Non- 72.39 72.79 69.24 71.47 1.40 85.86 89.49 80.98 85.44 1.17 0947- Vacci- 142 nated QWA- 51.9 Vacci- 83.85 74.96 78.42 79.07 1.26 89.70 83.02 81.24 84.65 1.18 0947- nated 143 QWA- 192.41 Vacci- 99.03 103.75 104.69 102.49 0.98 88.00 80.96 100.21 89.73 1.11 0947- nated 144 QWA- >1000 Vacci- 35.23 36.75 36.99 36.32 2.75 91.40 83.04 84.37 86.27 1.16 0947- nated 145 QWA- >1000 Vacci- BIG 3.21 3.17 3.47 3.28 30.45 4.66 4.45 3.82 4.31 23.18 0947- nated BLOOD 146 DRAW QWA- >1000 Vacci- 74.42 79.63 70.02 74.69 1.34 96.43 89.82 90.93 92.39 1.08 0947- nated 147 QWA- >1000 Vacci- 48.09 53.38 53.47 51.65 1.94 85.58 89.74 84.49 86.60 1.15 0947- nated 149 QWA- >1000 Vacci- 45.84 50.91 49.78 48.84 2.05 97.27 93.55 92.90 94.57 1.06 0947- nated 150 QWA- 24.03 Vacci- 76.16 78.93 83.53 79.54 1.26 104.49 103.12 97.68 101.76 0.98 0947- nated 151 QWA- 32.89 Vacci- 76.50 74.31 58.42 69.75 1.43 80.82 81.72 78.43 80.33 1.24 0947- nated 152 QWA- 0.36 Vacci- 92.38 95.06 83.56 90.33 1.11 81.13 82.29 70.79 78.07 1.28 0947- nated 153 QWA- 8.02 Vacci- 81.20 78.04 61.49 73.58 1.36 89.89 80.29 72.54 80.90 1.24 0947- nated 154 QWA- 271.68 Vacci- 83.98 86.99 69.06 80.01 1.25 79.14 80.93 74.47 78.18 1.28 0947- nated 155 QWA- >1000 Vacci- 23.65 20.82 15.97 20.15 4.96 78.25 75.10 58.66 70.67 1.42 0947- nated 156 QWA- 93.05 Vacci- 93.75 95.02 83.23 90.67 1.10 77.02 80.62 70.27 75.97 1.32 0947- nated 157 QWA- 261.09 Vacci- 78.75 78.50 64.50 73.92 1.35 88.22 86.79 71.09 82.03 1.22 0947- nated 158 QWA- 61.12 Vacci- 87.14 91.02 76.00 84.72 1.18 80.77 86.19 74.40 80.45 1.24 0947- nated 159 QWA- 381.69 Vacci- 72.70 68.79 62.46 67.98 1.47 81.13 74.64 66.11 73.96 1.35 0947- nated 160 QWA- 3.62 Vacci- 91.93 85.42 72.87 83.40 1.20 85.76 80.35 74.62 80.24 1.25 0947- nated 161 QWA- 198.58 Vacci- 91.35 92.25 79.20 87.60 1.14 112.63 106.14 95.29 104.69 0.96 0947- nated 162

SUPPLEMENTAL TABLE S2 Detailed information about cloned antibodies with paired heavy and light chains, Related to FIG. 2. Variable (V), diversity (D) and joining (J) genes, mutation on the variable gene (V MUT), and CDR3 amino acid sequences of cloned immunoglobulin heavy, kappa light and lambda light chains are listed. These antibodies are grouped by their IGHV genes, with our 5 selected H001-H020 antibodies indicated. H021 antibody used for sequence alignment in FIG. 2D is also indicated. The amino acid length of IGH CDR3 was between 5 and 27 amino acids, with the highest peak at 16 amino acids and the average around 15 amino acids. There are 16 of IGH CDR3 containing cysteines. HEAVY CHAIN KAPPA LIGHT CHAIN LAMBDA LIGHT CHAIN DONOR SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID ID V D J CDR 1 NO: CDR 2 NO: CDR 3 NO: V J CDR 1 NO: CDR 2 CDR 3 NO: V J CDR 1 NO: CDR 2 NO: CDR 3 NO: 9 IGH IGH IGHJ GYT 26 ISTY 27 ARN 28 IGKV IGKJ QSI 29 KAS QQY 30 V1- D6- 6*02 FTT NRNT GYS 1-5* 1*01 TNW YSY 18* 13* YG SSW 03 PWT 01 01 HGG THY YYY ALD F 55 IGH IGH IGHJ GYS 31 ISTY 32 ARD 33 IGL IGL SSD 34 EAS CSY 35 V1- D2- 4*02 FNT NGKT SVS V2- J3* VGS SGS 18* 15* YG WWN 23* 02 YDL TTC 01 01 LLF 01 V KSL EKL TLD Y 55 IGH IGH IGHJ GYS 36 ISTY 37 ARD 38 IGKV IGKJ QSV 39 GAS QQF 40 IGL IGL SSD 41 EVN NSY 42 V1- D2- 4*02 FNT NGKT SVS 3-20 4*01 SNS GSS V2- J1* IGG AGN 18* 15* YG WWN *01 Y PLT 8*0 01 YNY NNF 01 01 LLF 1 V KSL EKL TLD Y 55 IGH IGH IGHJ GYS 43 ISTY 44 ARD 45 IGKV IGKJ QSV 46 DAS QHR 47 V1- D2- 4*02 ENT NGKT SVS 3-11 1*01 SSY SNS 18* 15* YG WWN *01 WT 01 01 LLF KSL EKL TLD Y 55 IGH IGH IGHJ GYT 48 ISAH 49 ARD 50 IGL IGL ALP 51 KDS QSA 52 V1- D4- 4*02 FSS SGNT PDF V3- J3* VQF DST 18* 17* YG GDY 25* 02 ATY 01 01 GSD 02 WV IVD Y 99 IGH IGH IGHJ GYS 53 ISAY 54 ARW 55 IGL IGL SLR 56 GKN GSR 57 V1- D3- 6*03 FSS NGDT GVG V3- J3* TYY DNS 18* 16* YG MTF 19* 02 GYS 01 01 SYH 01 YHY MDV 99 IGH IGH IGHJ GYT 58 ISGY 59 ARG 60 IGKV IGKJ QSV 61 DAS QQY 62 V1- D6- 4*02 FGS SGKT RGD 3-11 4*01 GSY NDW 18* 13* FG SST *01 LT 01 01 WYS LY 146 IGH IGH IGHJ DYR 63 ISPF 64 AGD 65 IGKV IGKJ ESV 66 WAS QQY 67 V1- D1- 1*01 SIN NGNT TTS 4-1* 3*01 FFS YTT 18* 1* SG SAA 01 PHN PS 01 01 LTF RNY 146 IGH IGH IGHJ GYN 68 ISPY 69 ARF 70 IGKV IGKJ QSI 71 DAS QQY 72 V1- D1- 6*02 FIS NGKK FGG 1-5* 1*01 KTW NSY 18* 26* YA ATM 01 SGW 01 01 TVY T FYG LDV 146 IGH IGH IGHJ GYK 73 INAY 74 TRS 75 IGL IGL NSN 76 ANS ASW 77 V1- D6- 4*02 FTN NGHT EQW V1- J3* VGN DDS 18* 19* YG RSR 44* 02 NV LSG 01 01 GEY 01 SWV 146 IGH IGH IGHJ GYT 78 ISAS 79 GRD 80 IGKV IGKJ QSV 81 GAS QQY 82 V1- D1- 5*02 FSN SGNT DSG 3-20 4*01 SGS XSS 18* 26* YG SYP *01 Y PLA 01 01 MSP 146 IGH IGH IGHJ GYT 83 INAH 84 VRD 85 IGL IGL TGA 86 RTN LLY 87 V1- D1- 4*02 FRN NGDT INF V7- J3* VTS XWS 18* 20* YG IFD 43* 02 SYY SSA 01 01 Y 01 LG 69 IGH IGH IGHJ GYS 88 INVY 89 ARE 90 IGKV IGKJ LSV 91 DAS QQY 92 V1- D3- 6*03 FTS NANT GWF 3-15 1*01 SSN HEW 18* 10* YG GEF *01 PRT 04 01 RRN YNY NYY MDV 49 IGH IGH IGHJ GYT 93 FDPD 94 TLV 95 IGKV IGKJ QSV 96 AAS QQS 97 V1- D4- 3*01 LTE EGEV TRV 1-39 2*01 RTY YFA 24* 11* LS DAF *01 PYT 01 01 DV 49 IGH IGH IGHJ GYS 98 FDPD 99 TLV 100 IGKV IGKJ QNI 101 AAS QQS 102 V1- D4- 3*01 HTE EGET TRV 1-39 2*01 RNY YFA 24* 11* LP DAF *01 PYT 01 01 EV 49 IGH IGH IGHJ GYT 103 FDPD 104 TLV 105 IGKV IGKJ QNI 106 TAS QQS 107 V1- D2- 3*01 LTE EGET TGV 1-39 2*01 RTY YFA 24* 21* LP DAF *01 PYT 01 02 AV 49 IGH IGH IGHJ GYI 108 FDPD 109 TSV 110 IGKV IGKJ QNI 111 VAS QQS 112 V1- D3- 3*01 FSE EGET IKA 1-39 2*01 RTY YFA 24* 16* LS DAF *01 PYT 01 01 EV 13 IGH IGH IGHJ GYT 113 LNGG 114 ARG 115 IGKV IGKJ QSV 116 GAS QHY 117 IGL IGL NIR 118 ADS QVW 119 V1- D2- 6*02 FTH NDDR GWI 3-20 4*01 SSS NNP V3- J2* NKN DGG 3* 21* YA IQN *01 Q VA 21* 01 SYH 01 01 GGA 02 VI RYY HGM DV 13 IGH IGH IGHJ GYT 120 INAA 121 ARK 122 IGL IGL SSD 123 DVN CSY 124 V1- D3- 4*02 FTR NGDT DYY V2- J2* VGG AGN 3* 10* YP GSG 11* 01 YNY YIL 01 01 SYE 01 V FDN 69 IGH IGH IGHJ GYN 125 INVG 126 AKG 127 IGKV IGKJ ENI 128 KAS QQY 129 V1- D2- 3*01 FQR NGNT RSS 1-5* 2*01 GGW NSY 3* 2* SA HDL 03 SRY 01 01 YDP T FDF 99 IGH IGH IGHJ GYT 130 INPA 131 ARK 132 IGL IGL NSD 133 DVT SSY 134 V1- D3- 4*02 FTS   NGDT NYY V2- J3* VGG AGK 3* 10* YP ASG 11* 02 YNY YTL 01 01 SYH 01 V FDL 146 IGH IGH IGHJ GYT 135 INAG 136 ARV 137 IGKV IGKJ QSV 138 TAS QQS 139 V1- D1- 3*02 LTS SGLT GIP 1-39 4*01 STY SSV 3* 1* YA LRG *01 PLT 01 01 AGG SPF DI H001 146 IGH IGH IGHJ GYT 140 INAG 141 ARV 142 IGKV IGKJ QSI 143 SAS QQS 144 V1- D3- 3*02 FTT NGIT GIL 1-39 4*01 STY YST 3* 16* YA VRG *01 PLT 01 01 AGG SPF DI 146 IGH IGH IGHJ GYT 145 INAG 146 ARV 147 IGKV IGKJ QSI 148 TAS QQS 149 V1- D3- 3*02 FTT NGIT GIL 1-39 4*01 STY YST 3* 16* YA VRG *01 PLT 01 01 AGG SPF DI 146 IGH IGH IGHJ GYT 150 INIG 151 ARE 152 IGKV IGKJ QSV 153 KAS QLY 154 V1- D4- 3*01 FSR NGNT DYT 1-5* 4*01 STW NSY 3* 11* HA GNY 03 SGT 01 01 YDA T FDF 146 IGH IGH IGHJ GYS 155 INAA 156 ARD 157 IGL IGL SSN 158 GDT QSY 159 V1- D6- 6*02 FSN YGNT GVK V1- J2* IGK DSN 3* 13* YA EQL 40* 01 NYD LSG 01 01 VYY 01 SVV YFG MDV 146 IGH IGH IGHJ TYV 160 INAG 161 ARG 162 IGKV IGKJ QGI 163 AAS QKY 164 IGL IGL SSN 165 DNN GSW 166 V1- D3- 4*02 FTA NGDT ALL 1-27 3*01 SNY DSA V1- J1* IGN DSS 3* 10* YA WFR *01 PYT 51* 01 TY LYS 01 01 DDF 01 FYV DF 99 IGH IGH IGHJ GYT 167 INPS 168 ASR 169 IGKV IGKJ PNA 170 GAS QQY 171 V1- D2- 1*01 FSN GDST LDA 3-20 2*01 NSG GGL 46* 2* YH IPF *01 S PFT 01 01 QV 99 IGH IGH IGHJ GYI 172 IDPS 173 ARK 174 IGL IGL SSD 175 DVN SSY 176 V1- D4- 6*04 VTS DTYT GNY V2- J3* VGD TSS 46* 23* YR GSR 14* 02 YSY STG 01 01 YDW 01 V YFD V 55 IGH IGH IGHJ GYT 177 INPG 178 TRD 179 IGKV IGKJ QGI 180 AAS LQH 181 V1- D3- 3*01 FTN AGTT PIL 1-17 3*01 RND NGY 46* 9- FN RFY *01 PIT 03 01 DWQ SRD AFD V 60 IGH IGH IGHJ GGT 182 IVPI 183 ARV 184 IGKV IGKJ QSI 185 GAS QQS 186 IGL IGL SSD 187 EVT SSY 188 V1- D3- 6*01 FSS FGIP PSV 1-39 3*01 SNY YST V2* J1* VGG TGS 69* 3* YS ATC *01 LFS 14* 01 YKY STR 01 01 NFG 01 YV CYS AMD V 146 IGH IGH IGHJ GGK 189 TIPI 190 ARA 191 IGKV IGKJ QGI 192 GAS LQH 193 V1- D3- 4*02 FIA YGTA SFG 1-17 1*01 SNS NTY 69* 3* YG DLW *03 PWT 06 01 SGY PNQ FFD H 13 IGH IGH IGHJ GFS 194 IYGD 195 AHR 196 IGKV IGKJ QSI 197 KAS QQY 198 V2- D3- 5*02 FST GDE LLT 1-5* 1*01 SRW NSY 5* 9* GGV AYY 03 SWA 02 01 G DH 55 IGH IGH IGHJ GFS 199 IYWD 200 AHT 201 IGL IGL SSN 202 STN ATW 203 V2- D6- 5*02 LST DDK VAA V1- J2* IGS DDS 5* 13* FGV AAT 44* 01 NT LNG 02 01 G FWF 01 LV DP 69 IGH IGH IGHJ GFS 204 IYGD 205 AHS 206 IGL IGL ALP 207 EDN YST 208 V2- D2- 3*02 LST DDK SYF V3- J3* RKY DSS 5* 21* TAV DCG 10* 02 GDP 02 02 G GDC 01 V SDV AFD I 146 IGH IGH IGHJ GFS 209 IYWD 210 ARS 211 IGL IGL SSD 212 GVN CSY 213 V2- D2- 3*01 LTT DDK YCR V2- J2* VGG AGA 5* 15* NGM GGN 11* 01 YDY YTY 02 01 G CYS 01 VA TAF NV H002 146 IGH IGH IGHJ GFS 214 IDWD 215 ARS 216 IGL IGL NIG 217 DNS QVW 218 V2- D7- 4*02 LST DEK NHW V3- J1* GKT DTS 70 27* NTM GSH 21* 01 GDH D* 01 R FDY 02 LYV 04 55 IGH IGH IGHJ GFT 219 IRGS 220 ARD 221 IGKV IGKJ QSL 222 WAS QQF 223 V3- D2- 3*01 FSD HSSV LPG 4-1* 4*01 LYS YTA 11* 21* YY DEY 01 SNN PLT 01 01 LDA KNY FDL 60 IGH IGH IGHJ GLT 224 ISH 225 ASG 226 IGL IGL KLG 227 QDT QAW 228 V3- D6- 6*02 LSD DGS AAV V3- J2* DAY GSS 11* 13* YY TI PYF 1* 01 PAK 01 01 YYG 01 V VDV 99 IGH IGH IGHJ GFT 229 IGAA 230 ARA 231 IGL IGL SSN 232 SNN ATW 233 V3- D3- 4*02 FSS TDT VHY V1- J2* IGS DAS 13* 22* YD YDS 44* 01 NT LKG 01 01 SGH 01 VV YSG YYF DY 55 IGH IGH IGHJ GFT 234 IRSK 235 TTQ 236 IGL IGL NIG 237 YDS QVW 238 V3- D1- 4*02 FSN TDGG NAF V3- J1* SKS DSS 15* 1* AW TA ES 21* 01 SDH 01 01 01 YV 55 IGH IGH IGHJ GFT 239 IKSI 240 HTL 241 IGKV IGKJ QSV 242 GAS QQY 243 V3- D2/ 6*02 FSN TDGG STT 3-20 4*01 TSN INS 15* OR1 AY TI HYY *01 Y PLT 01 5- GMD 2a* V 01 146 IGH IGH IGHJ GFT 244 IQRK 245 AAH 246 IGKV IGKJ QSI 247 DAS QQY 248 V3- D6- 4*02 FSN TDGG NRA 1-5* 2*01 SNW YSY 15* 25* TY TA AY 01 SPL 01 01 T 9 IGH IGH IGHJ GLT 249 ISGS 250 ARA 251 IGL IGL SSN 252 DNN QSY 253 V3- D3- 4*02 FST SDYI RPP V1* J2* IGA DSS 21* 3* HS GTA 40* 01 GYD LSG 01 02 FGF 01 AL DH 13 IGH IGH IGHJ GFT 254 VSSS 255 VRT 256 IGKV IGKJ QSV 257 SAS QQY 258 V3- D3- 4*02 FSS SYSI FYF 3-15 1*01 RTN DIW 21* 16* YV DY *01 PPR 01 01 T 55 IGH IGH IGHJ GFT 259 ISSS 260 VRD 261 IGL IGL SSN 262 TNS AAW 263 V3- D4- 1*01 FSS SRYI MTT V1- J2* IGS DDS 21* 17* FS VTT 44* 01 HT LNG 01 01 CXX 01 LV QH 146 IGH IGH IGHJ GFT 264 ISSS 265 VRD 266 IGL IGL SSN 267 TNS AAW 268 V3- D4- 1*01 FSS SRYI MTT V1- J2* IGS DDS 21* 17* FS VTT 44* 01 HT LNG 01 01 CYL 01 LV QH 146 IGH IGH IGHJ GFS 274 ISSS 275 ARV 276 IGKV IGKJ QSV 277 SAS QQY 278 V3- D3- 4*02 FNA SSYI PIL 3-15 2*01 NSN SDW 21* 3* YS LAQ *01 PRY 01 01 GVP T TFD L 9 IGH IGH IGHJ GFT 279 VSGS 280 AKA 281 IGL IGL NIA 282 DDN QVW 283 V3- D2- 6*03 FTR GSST AIL V3- J2* SKS DSS 23* 2* YT GNY 21* 01 ADH 01 02 NYY 02 LVV MD V 13 IGH IGH IGHJ EFR 284 IIAT 285 VKD 286 IGL IGL NIG 287 DDN V3- D1- 4*01 FGS GAKT AIY V3- J2* SKS 23* 1* YA MSN 21* 01 01 01 WPW 02 YFD Y 13 IGH IGH IGHJ GFT 288 ISGS 289 AKD 290 IGL IGL NIG 291 EDS QVW 292 V3- D6- 4*02 FTN GGST PIY V3- J2* SRG DSS 23* 13* YA SSS 21* 01 SDH 01 01 WPY 02 PEV YFD V Y H021 13 IGH IGH IGHJ GFR 293 ISGS 294 AKD 295 IGKV IGKJ QSI 296 AAS QQS 297 IGL IGL NIG 298 DDS QVW 299 V3- D6- 4*02 FSS GGST PIY 1-39 2*03 SSY YSL V3- J2* SKS DSS 23* 13* YA TSR *01 YS 21* 01 SDH 01 01 WPY 02 SEV YFD I Y 13 IGH IGH IGHJ GFR 300 ISGR 301 AKD 302 IGL IGL NIG 303 DDT QVW 304 V3- D3- 4*02 FSS DAST GVL V3- J2* SKS DNS 23* 10* YA GSY 21* 01 SDH 01 01 HQY 02 PGV YFQ V Y 13 IGH IGH IGHJ GFT 305 ISGS 306 AKG 307 IGKV IGKJ QSV 308 GAF QHY 309 V3- DS- 4*02 FSS GGST SRN 3-15 4*01 SSN NHW 23* 12* YA GPY *01 SLT 01 01 IVA TLH FDY 55 IGH IGH IGHJ GFT 310 VSGN 311 VLS 312 IGL IGL SGI 313 YKS 314 MIW 315 V3- D6- 4*02 FNN GGST SSW V5- J3* NVG DSD HSS 23* 13* YA MDN 45* 02 TYR K AWV 01 01 PFD 02 F 55 IGH IGH IGHJ GFT 316 ASAS 317 AGF 318 IGKV IGKJ QSV 319 DAS QQR 320 V3- D1- 4*02 FSS GRNT PSG 3-11 1*01 SNH SNW 23* 26* YA THF *01 WT 01 01 FDY 55 IGH IGH IGHJ GFT 321 ASAS 322 AGF 323 IGKV IGKJ QSV 324 GAS QQF 325 IGL IGL SSD 326 EVN NSY 327 V3- D1- 4*02 FSS GRNT PSG 3-20 4*01 SNS GSS V2- J1* IGG AGN 23* 26* YA THF *01 Y PLT 8* 01 YNY NNF 01 01 FDY 01 V 55 IGH IGH IGHJ EFT 328 ISGS 329 ARP 330 IGL IGL SSN 331 SNN AAW 332 V3- D2- 6*02 FSS GDTT DAL V1- J3* IGT DDR 23* 2* YA HCS 44* 02 NT LIG 01 01 SIT 01 WV SCS LYG LAY YYG MDV 55 IGH IGH IGHJ GFT 333 ISGN 334 AKR 335 IGKV IGKJ QSV 336 GVS QQY 337 V3- D1- 4*02 FSS GGFT MVE 3-20 2*01 SNS GSS 23* 26* HG ATN *01 Y PPY 01 01 RYF T DY 55 IGH IGH IGHJ GFT 338 ISAN 339 ARD 340 IGL IGL SSD 341 DVN CSY 342 V3- D1- 4*02 FIN GIYT SSE V2- J2* VGG AGS 23* 26* YA WVL 11* 01 YNY YTV 01 01 GID 01 V F 60 IGH IGH IGHJ GFT 343 ITGS 344 AKD 345 IGL IGL NIG 346 DDT QVW 347 V3- D4/ 2*01 FSS GGST AVR V3- J2* IKS DSN 23* OR YA SAN 21* 01 SDH 01 15- HAW 02 PKV 4a* YFD V 01 F 60 IGH IGH IGHJ GFT 348 ISSN 349 AKG 350 IGKV IGKJ QSL 351 GAS QQY 352 V3- D5- 4*02 FSS GAGT YGL 3-15 1*01 VTN INW 23* 12* TA FDS *01 PPW 01 01 S H003 60 IGH IGH IGHJ GFT 353 LTAT 354 AKD 355 IGL IGL NIG 356 DDN QVW 357 V3- D2- 2*01 FSS GGNT AIR V3- J2* SKS DPT 23* 21* YA NSN 21* 01 SDQ 01 01 HAW 02 V YFD V 60 IGH IGH IGHJ GFT 358 ISGR 359 AKS 360 IGKV IGKJ QTI 361 AAS QQS 362 V3- D2- 4*02 FSS GDET RVT 1-39 5*01 GTY YST 23* 8* YG NSG *01 SIT 01 01 SID H 60 IGH IGH IGHJ GFR 363 ISGG 364 AKD 365 IGKV IGKJ QSV 366 WAS QQY 367 IGL IGL NIG 368 EDS QVW 369 V3- D4/ 2*01 FNN DGYT AIL 4-1* 4*01 LYS YST V3- J3* TNS DSS 23* OR YA SAN 01 SNN PLT 21* 02 SDH 01 15- HPW KNY 02 PKV 4a* YFD V 01 F 60 IGH IGH IGHJ GFT 370 IVNS 371 AKD 372 IGL IGL NIG 373 DDS QVW 374 V3- D2- 2*01 FSS GGST AIR V3- J2* SES DGS 23* 21* YA SSN 21* 01 SDH 01 01 HPW 02 PKV YFH L V 60 IGH IGH IGHJ GFR 375 ITGG 376 AKD 377 IGL IGL NIG 378 EDS QVW 379 V3- D4/ 2*01 FNN EGYT AIL V3- J3* TNS DRS 23* OR YA SAN 21* 02 SDQ 01 15- HPW 02 SKV 4a* YFD V 01 F 146 IGH IGH IGHJ GFT 380 ISGG 381 AKG 382 IGKV IGKJ QSV 383 GPS QQY 384 V3- D3/ 4*02 FST SEWS YGL 3-15 1*01 SSN INR 23* OR HA FDF *01 PPW 01 15- T 3a* 01 9 IGH IGH IGHJ GFT 385 IYTG 386 AKV 387 IGKV IGKJ QSV 388 DAS QQR 389 V3- D1- 4*02 FSN GSKT LLG 3-11 4*01 DSY STW 23* 1* YA GWN *01 PPS 03 01 GVF DH H004 146 IGH IGH IGHJ GFR 390 FSGS 391 AKD 392 IGL IGL NIG 393 EDS QVW 394 V3- D3- 6*02 FNN GSNI GYF V3- J2* SKS DSN 23* 10* YG GSG 21* 01 HDH 04 01 SLY 02 PGV GID V V 146 IGH IGH IGHJ GFT 395 ISGS 396 AKD 397 IGL IGL NIG 398 DDS QV 399 V3- D3- 6*02 FTS GGST GYY V3- J2* SKS WD 23* 10* YA GSG 21* 01 STS 04 01 SLY 02 DHP GMD GVV V 146 IGH IGH IGHJ GFT 400 FSG 401 AKV 402 IGL IGL SSN 403 DNN GTW 404 V3- D3- 6*02 FSS SGS IQY V1- J3* IGN DSS 23* 9* YA ST PRG 51* 02 NY LNN 04 01 FWF 01 CV YGM DV 60 IGH IGH IGHJ GFT 405 ISYD 406 ARD 407 IGL IGL SSD 408 EVS SSY 409 V3- D3- 6*02 FTS GSTH PGV V2- J2* VGG AGS 30- 10* YA PYY 8* 01 YHY NNY 3* 01 HYA 01 IL 01 MDV 146 IGH IGH IGHJ EFT 410 ISAD 411 VRD 412 IGKV IGKJ QGI 413 AAS LQH 414 IGL IGL GSN 415 SND STW 416 V3- D3- 4*02 FST GNNR ETD 1-17 2*01 RND NSY V1- J2* VGG DDS 30- 3* YA WEI *01 PRT 44* 01 NT LNG 3* 01 GVV 01 VV 01 VAT PEF DY 146 IGH IGH IGHJ GFP 417 LSFN 418 VTG 419 IGL IGL NKN 420 RND AAW 421 V3- D4- 4*02 FSS GDYI IRA V J2* VGN DSS 30- 23* HA RDY 10- 01 EG LSA 3* 01 GGS 54* MI 02 TFD 01 L 9 IGH IGH IGHJ GFR 422 IRYD 423 AKD 424 IGL IGL NIG 425 DDN QVW 426 V3- D3- 3*01 FTN GSKK GRW V3- J2* NTV ESS 30* 10* YG FGE 21* 01 TDP 02 01 SGG 02 VV FDV 9 IGH IGH IGHJ GFT 427 MSYD 428 ARD 429 IGKV IGKJ PSV 430 GVS QQY 431 V3- D2- 2*01 FTS GSYE YCS 3-20 4*01 SSS GSS 30* 2* YS RTN *01 Y PLT 03 01 CIN WIF DL 60 IGH IGH IGHJ GFR 432 ISND 433 AKD 434 IGL IGL NIG 435 DDR QVW 436 V3- D2- 6*02 FTG GSKK GYL V3- J2* GKS ETT 30* 8* YG SAA 21* 01 SDQ 03 02 RGY 02 LV GMD V 146 IGH IGH IGHJ GFT 437 ISYD 438 ARD 439 IGL IGL NSN 440 GNH QSY 441 V3- D4- 4*02 FSN GSNK TFG V1- J1* IGA DSR 30* 17* YD DYY 40* 01 GYD LSV 03 01 FDY 01 PYV 9 IGH IGH IGHJ KFT 442 TSYN 443 ARG 444 IGKV IGKJ QSV 445 WAS QQY 446 V3- D5- 6*02 FSK GGSK GGY 4-1* 3*01 LYS YST 30* 18* YA TYG 01 SNN PFT 04 01 SYY KNY YSM DV 9 IGH IGH IGHJ RFT 447 ISYD 448 ARG 449 IGKV IGKJ QSL 450 WAS QQY 451 V3- D5- 6*02 FSK GSSK GGY 4-1* 3*01 LYS YST 30* 18* YA TYG 01 SNN PFT 04 01 SYY KNY YAM DV 55 IGH IGH IGHJ GFT 452 MSNT 453 ARA 454 IGKV IGKJ QSV 455 DAS QHR 456 V3- D1- 4*02 FSS GSTK LLS 3-11 1*01 SSY SNS 30* 26* YS VVG *01 WT 04 01 SKS YYF DF 55 IGH IGH IGHJ GFT 457 MSNT 458 ARA 459 IGKV IGKJ QSV 460 DAS QHR 461 V3- D1- 4*02 FSS GSTK LLS 3-11 1*01 SSY SNS 30* 26* YS VVG *01 WT 04 01 SKS YYF DF 55 IGH IGH IGHJ GFN 462 ISYD 463 ARD 464 IGL IGL NSN 465 NSD GTW 466 V3- D1- 6*02 FNV GSKK EKY V1- J2* IGN DSS 30* 26* YA SGL 51* 01 NF LSL 04 01 YSG 01 GV RTG DYY YGM DV 55 IGH IGH IGHJ GFT 467 ISYD 468 ARD 469 IGL IGL SSD 470 DVN SSY 471 V3- D3- 6*02 FSA GSNR GKL V2- J2* VGG TSS 30* 22* YS GRT 14* 01 YNY TSL 04 01 YHD 01 V SRQ SYF YIM DV 13 IGH IGH IGHJ GFR 472 TSFD 473 AKD 474 IGL IGL NIG 475 DDN QVW 476 V3- D3- 6*02 FSS GSKT AYY V3- J2* SKS GSG 30* 10* YG FAS 21* 01 GVI 18 01 GSF 02 FGM DV 13 IGH IGH IGHJ GFT 477 ISYD 478 AKD 479 IGKV IGKJ QSI 480 KAS QQY 481 IGL IGL SSN 482 RNN AAW 483 V3- D3- 6*02 FSR GSNK AYY 1-5* 4*01 SVW TSF V1- J1* IGS DDR 30* 10* YG YGS 03 ST 47* 01 DY LSG 18 01 GYG 01 YV MDV 60 IGH IGH IGHJ GFT 484 ISSD 485 AKD 486 IGL IGL NIG 487 DDS QVW 488 V3- D3- 6*02 FRS GSKK GYV V3- J2* SKS DSS 30* 10* YG VSG 21* 01 SDH 18 01 SGY 02 VV GMD V H009 60 IGH IGH IGHJ RFS 489 ISYD 490 AKT 491 IGL IGL NIG 492 DDN QVW 493 V3- D1- 6*02 FNT GSHE DIK V3- J2* RKS DGT 30* 26* YG WGA 21* 01 RDH 18 01 TNY 02 LVV GMD V 60 IGH IGH IGHJ GFT 494 TLYD 495 AKD 496 IGKV IGKJ QGI 497 AAS LQH 498 IGL IGL SLR 499 NKD NSR 500 V3- D5- 4*02 FSN GSHS SAG 1-17 1*01 RTD NSY V3- J2* SFY DSI 30* 12* YA YGL *01 PWT 19* 01 GNH 18 01 HY 01 VV 60 IGH IGH IGHJ GFR 501 ISND 502 AKD 503 IGL IGL NVG 504 DDS QVW 505 V3- D3- 6*02 FTG GSKK GYL V3- J2* SKS DTT 30* 22* YG SAA 21* 01 TDQ 18 01 RGY 02 LV GMD V 60 IGH IGH IGHJ GFR 506 ISND 507 AKD 508 IGL IGL NIG 509 DDS QVW 510 V3- D3- 6*02 FTG GSKT GYL V3- J2* GKS DTT 30* 22* YG SAA 21* 01 SDQ 18 01 RGY 02 LV GMD V 60 IGH IGH IGHJ GFR 511 ISND 512 AKD 513 IGL IGL NIG 514 DDN QVW 515 V3- D3- 6*02 FTG GSKK GYL V3- J2* ALS DTS 30* 22* YG SAA 21* 01 SDQ 18 01 RGY 02 LV GMD V H005 60 IGH IGH IGHJ GFR 516 ISND 517 AKD 518 IGL IGL NIG 519 DDS QVW 520 V3- D3- 6*02 FTG GSKK AYL V3- J2* GKS DTA 30* 16* YG SAA 21* 01 SDQ 18 01 RGY 02 LV GMH V 60 IGH IGH IGHJ GFR 521 ISND 522 AKD 523 IGL IGL NIG 524 DDR QVW 525 V3- D3- 6*02 FTG GSKK GYL V3- J2* GKS DTT 30* 22* YG SAA 21* 01 SDQ 18 01 RGY 02 LV GMD V 60 IGH IGH IGHJ GFR 526 ISND 527 AKD 528 IGL IGL NIG 529 DDT QVW 530 V3- D3- 6*02 FTG GSKK GYL V3- J2* GKS DTT 30* 22* YG SAA 21* 01 SDQ 18 01 RGY 02 LV GMD V 60 IGH IGH IGHJ GFR 531 ISND 532 AKD 533 IGL IGL NIG 534 DDS QVW 535 V3- D3- 6*02 FTG GSKK GYL V3- J2* GKS DTT 30* 22* YG SAA 21* 01 SDQ 18 01 RGY 02 LV GMD V 60 IGH IGH IGHJ GFT 536 ISYD 537 AKT 538 IGL IGL NIG 539 DDN QVW 540 V3- D1- 6*02 FNT GSNK DIR V3- J2* SKS DGS 30* 26* YA WGA 21* 01 SDH 18 01 TNY 02 LVV GMD V H010 60 IGH IGH IGHJ GFS 541 ISYD 542 AKG 543 IGL IGL NIG 544 DDS QVW 545 V3- D3- 4*02 FST GMIK PLF V3- J2* DMS DNS 30* 3* YG GLF 21* 01 RNR 18 01 SFD 03 GI Q 60 IGH IGH IGHJ GFR 546 ISND 547 AKD 548 IGL IGL NIG 549 DDR QVW 550 V3- D3- 6*02 FTG GSKK GYL V3- J2* GKS DTT 30* 22* YG SAA 21* 01 SDQ 18 01 RGY 02 LV GMD V 60 IGH IGH IGHJ GFR 551 ISND 552 AKD 553 IGKV IGKJ QSL 554 KVS TQV 555 V3- D3- 6*02 FTG GSKR GYL 2-30 1*01 VYS TLW 30* 22* YS SAA *01 DGN PPW 18 01 RGY TY T GMD V H006 60 IGH IGH IGHJ GFR 556 ISND 557 AKD 558 IGL IGL NIG 559 DDN QVW 560 V3- D3- 6*02 FTG GSKK GYL V3- J2* GKS DTT 30* 22* YG SAA 21* 01 SDQ 18 01 RGY 02 LV GMD V 60 IGH IGH IGHJ GFR 561 ISND 562 AKD 563 IGL IGL SSD 564 EVS SSY 565 V3- D3- 6*02 FTG GSKK GYL V2- J2* VGS TSS 30* 22* YG SAA 18* 01 YNR STL 18 01 RGY 02 V GMD V 60 IGH IGH IGHJ GFT 566 ISSD 567 AKD 568 IGL IGL NIG 569 DDT QVW 570 V3- D2- 4*02 FRS GSKK PIK V3- J2* SKS DSN 30* 21* YG VSA 21* 01 SDH 18 02 NGW 03 VV GFD Y 60 IGH IGH IGHJ GFR 571 ISND 572 AKD 573 IGL IGL NIG 574 DDS QVW 575 V3- D3- 6*02 FTG GSRK GYL V3- J2* GKS DTT 30* 22* YG SAA 21* 01 SDQ 18 01 RGF 02 LV GMD V 60 IGH IGH IGHJ RFS 576 ISYD 577 AKT 578 IGL IGL NIG 579 DDN QVW 580 V3- D2- 6*02 FST GSEK DIM V3- J2* SKS DDS 30* 15* YG WRA 21* 01 RDH 18 01 VNY 02 LVI GMD V 60 IGH IGH IGHJ RFS 581 ISYD 582 AKT 583 IGL IGL NIG 584 DDN QVW 585 V3- D2- 6*02 FST GSEK DIM V3- J2* SKS DDS 30* 15* YG WRA 21* 01 RDH 18 01 VNY 02 LVI GMD V 60 IGH IGH IGHJ GFS 586 ISYD 587 AKT 588 IGL IGL NIG 589 DDN QVW 590 V3- D2- 6*02 FST GSSK DIM V3- J2* SKS DDS 30* 21* YG WQA 21* 01 RDH 18 01 VNY 02 LVI GMD V 60 IGH IGH IGHJ GFR 591 ISND 592 AKD 593 IGL IGL NIG 594 DDR QVW 595 V3- D3- 6*02 FTG GSKK GYL V3- J2* GKS DTT 30* 22* YG SAA 21* 01 SDQ 18 01 RGY 02 LV GMD V 60 IGH IGH IGHJ RFS 596 ISYD 597 AKT 598 IGL IGL NIG 599 DDN QVW 600 V3- D2- 6*02 FST GSEK DIM V3- J2* SKS DES 30* 15* YG WRA 21* 01 RDH 18 01 VNY 02 LVI GMD V 60 IGH IGH IGHJ GFR 601 ISND 602 AKD 603 IGL IGL NIG 604 DDR QVW 605 V3- D3- 6*02 FTG GSKK GYL V3- J2* GKS DTT 30* 22* YG SAA 21* 01 SDQ 18 01 RGY 02 LV GMD V H008 146 IGH IGH IGHJ GFR 606 IPFD 607 AKD 608 IGL IGL DVG 609 DDT QVW 610 V3- D1- 6*02 FTM GRTQ GIL V3- J2* SKS DSS 30* 26* YG GAR 21* 01 SDH 18 01 RGL 02 VV YGI DV H007 146 IGH IGH IGHJ GFT 611 ISYD 612 AKE 613 IGK IGKJ QTI 614 GAS QQY 615 V3- D3- 6*02 FNN GSNK IGG V3- 2*01 YTT SSS 30* 3* YA FDF 20* Y PPG 18 01 RSG 01 YT SQR SYY YYG VDV 146 IGH IGH IGHJ GFT 616 ISYD 617 AKE 618 IGKV IGKJ QSV 619 GAS HQY 620 V3- D3- 6*02 FSR GGNK IGG 3-20 2*01 YST VTS 30* 3* HG FDF *01 Y PPG 18 01 RSG YT DQL TYY YYG MDV 146 IGH IGH IGHJ GFS 621 ISYD 622 AKD 623 IGL IGL NIA 624 DDT QVW 625 V3- D5- 6*02 FSN GSNK AYI V3- J2* SKS DSS 30* 18* YG YAR 21* 01 SND 18 01 GSY 02 PVV YGM DV 146 IGH IGH IGHJ GFR 626 ISFD 627 AKD 628 IGL IGL NIR 629 DDN QVW 630 V3- D1- 6*02 FTK GSTQ GIL V3- J2* SKN DSY 30* 26* YG GAR 21* 01 SDH 18 01 RGX 02 VV YGI DV 146 IGH IGH IGHJ GFT 631 ISFD 632 AKE 633 IGKV IGKJ QTV 634 GAS QQY 635 V3- D3- 6*02 FSN GSNK IGG 3-20 2*01 YNT GNS 30* 3* YA FDF *01 Y PPG 18 01 RSG YT KQR SYY YYG VDV 146 IGH IGH IGHJ GFR 636 VSYD 637 AKD 638 IGL IGL NIG 639 DDS QVW 640 V3- D3- 4*02 FTI GSKQ AYY V3- J3* SQS DSS 30* 10* YG YGS 21* 02 SMG 18 01 GSH 02 V NNP DY 146 IGH IGH IGHJ GFT 641 ISYD 642 AKG 643 IGL IGL NSN 644 ANS ASW 645 V3- D3- 4*02 FSN GRDK YDY V1- J3* VGN DDS 30* 16* YG IWG 44* 02 NV LSG 18 02 TYR 01 SWV PRP DLD S 146 IGH IGH IGHJ GFT 646 VSYD 647 AKD 648 IGL IGL NIG 649 DDR QVW 650 V3- D6- 4*02 FSD GTSE PVQ V3- J2* SKT HST 30* 13* YG RSN 21* 01 TEP 18 01 WYY 02 VV FDY 146 IGH IGH IGHJ GFT 651 TSYD 652 AKD 653 IGL IGL YIG 654 DDR QVW 655 V3- D1- 4*02 FSN GINK PVH V3- J2* SKT YSN 30* 20* YG RSN 21* 01 SEP 18 01 WFY 02 VV FDH 146 IGH IGH IGHJ GTS 656 ISPN 657 AKG 658 IGKV IGKJ QSI 659 KAS QYY 660 IGL IGL NIG 661 DDN QVW 662 V3- D3- 6*02 FST AFDK SPI 1-5* 1*01 DTW SVY V3- J3* SKN DNN 30* 3* SG IRF 03 ST 21* 02 SDH 18 01 LMM 02 VV DV 13 IGH IGH IGHJ GFT 663 IWSD 664 ARE 665 IGL IGL NIR 666 ADS QVW 667 V3- D6- 4*02 FSS GSNK AGI V3- J2* GKS DSS 33* 13* YG AAP 21* 01 SDH 01 01 ASL 02 VV DF 13 IGH IGH IGHJ GFT 668 IWSD 669 TRE 670 IGL IGL NIG 671 DDN QVW 672 V3- D6- 4*02 FSN GTNK AGI V3- J2* NKN DSS 33* 13* YG AAP 21* 01 SYH 01 01 AAL 02 VV DY H011 13 IGH IGH IGHJ GFT 673 VWYD 674 VRD 675 IGKV IGKJ QSI 676 GTS QHR 677 V3- D1- 3*01 FSN GSYK NWS 3-11 2*03 SSY NSW 33* 7* YG YNA *01 PYS 01 01 FDV 13 IGH IGH IGHJ GFI 678 IWKD 679 VRE 680 IGKV IGKJ QGI 681 TAS LQH 682 V3- D6- 4*02 FSD GSNK NSG 1-17 4*01 RNN DSY 33* 19* YG WYY *01 PFT 01 01 FDY H012 13 IGH IGH IGHJ GFA 683 IWHD 684 ARE 685 IGL IGL NIR 686 ADS QVW 687 V3- D6- 4*02 FRS GSNK GAI V3- J2* SRN DSG 33* 13* YG AAP 21* 01 TDH 01 01 ASL 02 VI DV 13 IGH IGH IGHJ GFT 688 IWSD 689 ARE 690 IGL IGL NIR 691 ADS QVW 692 V3- D6- 4*02 FSS GSNQ GGI V3- J2* NKN DGG 33* 13* FG AAP 21* 01 SYH 01 01 AAL 02 VI DF 13 IGH IGHJ GVT 693 IWYD 694 ARE 695 IGKV IGKJ QNI 696 DAS QHS 697 V3- 4*02 FNS GTNK SKA 1-39 2*03 SIF SFP 33* YG YPY *01 PQD 01 YFD S Y 13 IGH IGH IGHJ GFT 698 IWYD 699 ARE 700 IGKV IGKJ QGI 701 AAS LQH 702 V3- D1- 4*02 FSN GTYK SNG 1-17 1*01 RNN NSF 33* 1* YA FGS *01 PRT 01 01 DF 13 IGH IGH IGHJ GFV 703 VWYD 704 VRD 705 IGKV IGKJ QSI 706 ATS QHR 707 V3- D3- 3*02 FSS GSYK NWS 3-11 2*03 SSY NSW 33* 10* YG YNA *01 PYS 01 01 FDI 13 IGH IGH IGHJ GFT 708 IWYD 709 ARD 710 IGKV IGKJ QSV 711 DAS QHR 712 V3- D1- 3*02 FSN GSYK NWK 3-11 2*03 SSY SNW 33* 20* YG YNA *01 PYS 01 01 FDI 13 IGH IGH IGHJ GFT 713 IWSD 714 ARE 715 IGL IGL SSD 716 EGS CLY 717 V3- D6- 4*02 FSR GSNQ SSG V2- J1* VGN AGS 33* 19* YG WYY 23* 01 YNF SIS 01 01 FDY 01 YV 13 IGH IGH IGHJ GFT 718 IWYD 719 ARE 720 IGL IGL NIG 721 ADS QVW 722 V3- D6- 5*02 FSS GSNE ERI V3- J2* RKN DSS 33* 13* FG AAP 21* 01 TYH 01 01 ASL 02 VV DL 13 IGH IGH IGHJ GFT 723 IWHD 724 ARE 725 IGL IGL NIA 726 ADS QVW 727 V3- D6- 4*02 FSS GTNQ LRI V3- J2* NKN DSG 33* 25* YG AAP 21* 01 SDH 01 01 AAL 02 VL DY 49 IGH IGH IGHJ GFT 728 IWYD 729 ARQ 730 IGKV IGKJ QGV 731 DAS QHY 732 V3- D3- 4*02 FSS GSHK MFT 3-15 1*01 SSN NNW 33* 16* FN GHF *01 PRT 01 01 DY 60 IGH IGH IGHJ GFT 733 IWND 734 ARE 735 IGKV IGKJ QSV 736 GAS QQY 737 V3- D2- 6*02 FSG GSFK GRG 3-20 3*01 SSS GRS 33* 2* YG QLL *01 Y QGF 01 01 FHG T MDV 60 IGH IGH IGHJ GFT 738 IWSS 739 ARD 740 IGL IGL SSD 741 EGT CSF 742 V3- D2- 4*02 FSG GSKT GHC V2- J3* VGN AGS 33* 21* HG DGG 23* 02 YNL RWV 01 02 CYS 01 ALY DY H015 60 IGH IGH IGHJ GFS 743 IWFD 744 ARE 745 IGKV IGKJ QGL 746 SAS QQS 747 V3- D6- 4*02 FSR GTND DPH 1-39 4*01 TSF YGT 33* 6* HG LLI *01 PAL 01 01 ATL A DL 60 IGH IGH IGHJ GFT 748 IWAD 749 ARE 750 IGKV IGKJ QSI 751 TAS QQS 752 V3- D4- 2*01 FRS GTKQ TTI 1-39 4*01 NKY FSI 33* 11* YG FNW *01 PPT 01 01 YFD L 60 IGH IGH IGHJ GFT 753 IWSD 754 ARE 755 IGL IGL SSD 756 DVS CSY 757 V3- DS- 4*02 FSD GSNK RRG V2- J2* VGG AGR 33* 18* YG FSY 11* 01 YNS YTF 01 01 GLD 01 VV DN 60 IGH IGH IGHJ GFT 758 IWKD 759 ARE 760 IGKV IGKJ QRI 761 AAS QQA 762 V3- D6- 3*01 FSR GTND QAE 1-39 4*01 GDF YNA 33* 19* YG IAV *01 PPL 01 01 ASF T DF 60 IGH IGH IGHJ GFT 763 IWKD 764 ARE 765 IGL IGL RSN 766 GND GSW 767 V3- D6- 4*02 FSN GTNK SHY V1- J3* IGS DGS 33* 19* YG SAW 51* 02 NY LSV 01 01 YVL 01 GV DY 60 IGH IGH IGHJ GFT 768 IWYD 769 ARE 770 IGKV IGKJ QTI 771 ATS QQS 772 V3- D2- 4*02 FSR GSNK DPN 1-39 4*01 TRS DST 33* 8* HG VFI *01 PAL 01 01 ATL A DL 60 IGH IGH IGHJ GFT 773 IWND 774 ARE 775 IGL IGL SSD 776 DVS CSY 777 V3- D3- 4*02 FSR GSTK DPY V2- J3* VGG AGS 33* 16* YG VFM 11* 02 YNY YTW 01 01 ATL 01 V DS 60 IGH IGH IGHJ GFT 778 IWAD 779 ARE 780 IGKV IGKJ QSI 781 GAS QQS 782 V3- D2- 2*01 FRN GTNQ TTI 1-39 4*01 NNY FSI 33* 21* YG FQW *01 PPT 01 01 YFD L 69 IGH IGH IGHJ GFT 783 IWYD 784 ARE 785 IGKV IGKJ QSL 786 QIS MQA 787 V3- D2- 6*02 FSS GSLK TTF 2-24 1*01 VHS AQF 33* 15* YG GRF *01 DGN PWT 01 01 CSG TY GSC YSD YYY GMD V 69 IGH IGH IGHJ GFV 788 IWAD 789 ARE 790 IGL IGL SSN 791 DNN V3- D6- 4*02 FSN GTNS GGI V1- J1* IGN 33* 13* YG VAA 51* 01 NY 01 01 DK 01 H014 146 IGH IGH IGHJ GFS 792 IWRD 793 ARE 794 IGL IGL KIV 795 DDD QVW 796 V3- D4/ 4*02 FSD GSNS ARV V3- J3* NKN DNG 33* OR YG AAP 21* 02 SNH 01 15- ASY 02 VV 4a* DY 01 146 IGH IGH IGHJ GFT 797 IWAD 798 ARE 799 IGL IGL NIR 800 DDD QVW 801 V3- D6- 4*02 FSS GTNK ALI V3- J2* SKN DNN 33* 13* CG AAP 21* 01 SRH 01 01 ATF 02 VV DY 146 IGH IGH IGHJ GFT 802 IWAD 803 ARE 804 IGL IGL NIR 805 DDD QVW 806 V3- D6- 5*02 FRN GSNK GHI V3- J2* NKN DSS 33* 13* YG AAP 21* 01 SEH 01 01 AAL 02 VV DL H013 146 IGH IGH IGHJ GFT 807 IWSD 808 ARE 809 IGL IGL NIR 810 DDN QVW 811 V3- D6- 4*02 FSG GSNK ANI V3- J2* SKN DSY 33* 13* NG AAP 21* 01 SDH 01 01 AIY 02 VV DH 146 IGH IGH IGHJ GFT 812 IWAD 813 ARE 814 IGKV IGKJ QSI 815 VAS QQS 816 V3- D2- 5*02 FTT GSNQ GHV 1-39 4*01 ANY YSM 33* 15* YG ATP *01 PTL 01 01 ILD  T L 146 IGH IGH IGHJ GFT 817 IWYD 818 VRD 819 IGKV IGKJ QSV 820 EAT QHR 821 V3- D3- 3*01 FSS GSIK NFG 3-11 2*01 TRY SNW 33* 10* YG LNA *01 PYT 01 01 FDV 146 IGH IGH IGHJ GFT 822 IWHD 823 ATE 824 IGL IGL SGI 825 SXS 826 MIX 827 V3- D6- 4*02 FSN GSNQ RRI V5- J2* SVD DK HSS 33* 13* YG AAP 48* 01 RSR AMW 01 01 GCL 02 DY 146 IGH IGH IGHJ GFT 828 IWYD 829 ARE 830 IGKV IGKJ QSI 831 KAS QYY 832 IGL IGL NIG 833 DDN QVW 834 V3- D6- 4*02 FSS GSTK ALI 1-5* 1*01 DTW SVY V3- J3* SKN DNN 33* 13* HG AAP 03 ST 21* 02 SDH 01 01 ATF 02 VV DY 60 IGH IGH IGHJ GFS 835 IWYD 836 AGG 837 IGKV IGKJ QDI 838 DAS QQY 839 IGL IGL EDN YST 840 V3- D5- 6*02 FSR GSTR GYS 1-33 4*01 SNY DNL V3- J2* DRS 33* 12* YG SRG *01 PPL 10* 01 GDQ 02 01 YYN T 01 RV YGL DV 99 IGH IGH IGHJ GFT 841 IWSD 842 AKA 843 IGL IGL SSN 844 GNS QSY 845 V3- D2- 4*02 FSR GSNK TCG V1- J3* IGA DSN 33* 15* YG DGS 40* 02 GYD LSG 06 01 CGL 01 WV YYF DY 55 IGH IGHD IGHJ INGN 846 AKD 847 IGKV IGKJ QSL 848 KVS MQQ 849 V3- 2-21 5*02 GRDT IWI 2-30 1*01 VYS THW 43* *01 FDG *01 DGN PWA 02 RRW TY IAG SPD A 49 IGH IGH IGHJ GFS 850 ITSN 851 ARA 852 IGKV IGKJ QSL 853 WAS QQY 854 V3- D1- 6*02 FSS SATI GPP 4-1* 4*01 LYR YTA 48* 1* YS SPP 01 SNN PLL 01 01 NYG KNY MDV H016 69 IGH IGH IGHJ GFT 855 ISTT 856 ASV 857 IGKV IGKJ QSI 858 RAS QQY 859 V3- D3/ 2*01 FPS SEAI GLD 3-15 4*01 SSN DHW 48* OR HT SKI *01 PLT 02 15- SGY 3a* WYF 01 DL 69 IGH IGH IGHJ GFT 860 ISSS 861 ARV 862 IGKV IGKJ QSV 863 GAS QQY 864 V3- D3/ 2*01 FST GDTI GLA 3-15 4*01 SSN NDW 48* OR YT LTI *01 PLT 02 15- SGY 3a* WYF 01 DL 146 IGH IGH IGHJ GFT 865 ISTT 866 ARA 867 IGKV IGKJ QSV 868 AAS QQY 869 V3- D7- 2*01 FSS SAAI KLG 3-15 4*01 GSN NNW 48* 27* SV SGS *01 PLT 02 01 YWY FDL 13 IGH IGH IGHJ GIT 870 ISSD 871 ARD 872 IGL IGL SGI 873 YRS 874 MIW 875 V3- D1- 3*02 LRT DKTI TGI V5- J1* NVG DSD HST 48* 1* YK WNG 45* 01 TYR M AYV 03 01 AYD 02 AFD I 13 IGH IGHJ GFT 876 ISNS 877 VGF 878 IGKV IGKJ RSL 879 WAS QQY 880 V3- 4*02 FSS GNTI DH 4-1* 5*01 LYT YSP 48* YE 01 SVN PIT 03 KNH 55 IGH IGH IGHJ GFT 881 ISSS 882 AGH 883 IGL IGL SSN 884 RNS QSY 885 V3- D2- 4*02 FSN GSVM CSS V1- J2* IGA DSS 48* 2* NE NKC 40* 01 GYD LSG 03 02 YKY 01 SI 69 IGH IGH IGHJ GFT 886 IKPK 887 ARD 888 IGKV IGKJ QSL 889 WAS QQY 890 V3- D3- 4*02 FGD AYGG LTI 4-1* 5*01 LYS YST 49* 22* YG AT NKI 01 SNN PIT 03 01 IVA KNY NDF 146 IGH IGH IGHJ GFT 891 IRSK 892 ARV 893 IGKV IGKJ QSV 894 WAS QQY 895 V3- D6- 4*02 FGD GYGG PYS 4-1* 4*01 LYS YST 49* 13* YA TR SSW 01 FNN PLT 03 01 YVA KNY WAD Y 99 IGH IGH IGHJ GFT 896 IRRK 897 TRG 898 IGKV IGKJ QSL 899 ELS MQS 900 V3- D3- 4*02 FGD ANRG DYY 2D- 1*01 LYS IQL 49* 10* YG TT GSR 29* DGK RT 04 01 NSY 01 TY FWL FDY 69 IGH IGH IGHJ GFT 901 IYSG 902 ARV 903 IGL IGL SGT 904 STT LLY 905 V3- D6- 4*02 VIS VNT IAV V7- J3* VTT CSG 53* 19* NY AGT 43* 02 ANY VRV 01 01 NRG 01 GPR WRS TYY FDY 9 IGH IGH IGHJ GLT 906 INEE 907 ASE 908 IGL IGL SSN 909 DSY GTW 910 V3- D3- 4*02 FSR GSHS LWT V1- J3* VGK DSS 7* 3* YW AFN 51* 02 NY LKV 01 01 KDW 01 VV SGY NDY 9 IGH IGH IGHJ GFT 911 IKQD 912 TRD 913 IGKV IGKJ QGI 914 ASS LQH 915 V3- D1- 4*02 FTN GSEK TWV 1-17 3*01 SKY QSY 7* 26* FK DS *03 PFT 01 01 13 IGH IGH IGHJ GFS 916 IKED 917 ASS 918 IG IGL NKN 919 RHN SAW 920 V3- D2- 4*02 FSN GSEK HYS LV J3* VGN DFS 7* 21* YW AGD 10- 02 KG LRA 01 01 VSY 54* WV NFD 01 Y 55 IGH IGH IGHJ GFI 921 IKQD 922 ARS 923 IGKV IGKJ QSV 924 DAS QHR 925 V3- D6- 5*02 FSS GSDK HVA 3-11 2*01 SSY SK 7* 13* SW AGV *01 03 01 TRW FDP 55 IGH IGH IGHJ SFT 926 INQD 927 ARS 928 IGKV IGKJ QNI 929 DAS HHR 930 V3- D6- 5*01 FST GSER HVA 3-11 2*01 NSQ IN 7* 13* SW AGG *01 03 01 TRW IDS 55 IGH IGH IGHJ GFT 931 INQD 932 ARL 933 IGL IGL SSN 934 INN AAW 935 V3- D3- 3*01 FSD GSEY DRG V1- J2* IGS DDS 7* 16* YVV TGE 44* 01 RS LNG 03 02 SGY 01 VV RSS DV 55 IGH IGH IGHJ GFI 936 INQD 937 ARS 938 IGKV IGKJ QSV 939 DAS QHR 940 V3- D1- 5*02 FSS GSDI HVA 3-11 2*01 SSY SY 7* 7* NW ASG *01 03 01 TRW FDP 55 IGH IGH IGHJ GFT 941 TRNK 942 ARD 943 IGKV IGKJ QSV 944 DAS QHR 945 V3- D3- 4*02 FSD ANSY GYD 3-11 2*01 SSY SY 72* 9* HF TT ILN *01 01 01 HFV RFD F 60 IGH IGH IGHJ GFT 946 IRNK 947 ARE 948 IGL IGL SSD 949 EVT SSY 950 V3- D3- 3*02 FSD AKSY GLG V2- J3* VGG TTS 72* 16* HY TT SPT 14* 02 YNY STL 01 01 SDA 01 V FDI 60 IGH IGH IGHJ GFT 951 IRSK 952 TSQ 953 IGKV IGKJ QNI 954 GAS QHY 955 V3- D4- 6*02 LSG ANNY YGD 3-15 3*01 RNN NNW 73* 17* SA AT GYY *01 PLF 02 01 YAM T DV 9 IGH IGHJ ISDD 956 VRG 957 IGL IGL NSD 958 DVT CSF 959 V3- 6*02 ERST LNH V2- J3* VGG TTR 74* AMD 14* 02 YNF NTW 01 V 01 V 13 IGH IGH IGHJ GFT 960 IKYD 961 ARV 962 IGKV IGKJ QSL 963 WAS QQY 964 V3- D3- 4*02 FST GSST YRD 4-1* 4*01 LYS YDI 74* 22* YR SRD 01 SNK PYT 01 01 GSD KNY FRH FDS H017 55 IGH IGH IGHJ GFT 965 ISTD 966 ARG 967 IGL IGL SSD 968 DVT SSY 969 V3- D3- 4*02 FSN GSST STY V2- J1* IGV RGS 74* 10* YW YFG 14* 01 YNY STP 01 01 SGS 01 YV VDY 55 IGH IGH IGHJ GFT 970 IESD 971 ARG 972 IGL IGL RSD 973 DVS YSY 974 V3- D1- 4*02 FSD GSGT SLD V2- J2* VGA TTS 74* 26* YW F 14* 01 YNY NTL 01 01 01 V 55 IGH IGH IGHJ GFT 975 IDDG 976 SRG 977 IGL IGL RSD 978 DVS YSY 979 V3- D1- 4*02 FSD GSAT SLD V2- J2* VGA TTS 74* 26* YW Y 14* 01 YNY NTL 01 01 01 V 99 IGH IGH IGHJ GFT 980 INSD 981 ACL 982 IGL IGL TGA 983 DAS LLS 984 V3- D4/ 4*02 FSN GTNT RVP V7- J2* VTS YSG 74* OR YW DRN 46* 01 GHY AQV 01 15- 01 4a* 01 13 IGH IGH IGHJ GFI 985 ISWN 986 VKA 987 IGKV IGKJ QSV 988 DAS QQY 989 V3- D2- 4*02 FDD SEFM NVK 3-20 1*01 SSS SGS 9* 2* YS KGS *01 Y SPR 01 01 TSC T FDY 60 IGH IGH IGHJ GFN 990 ISYN 991 VKD 992 IGL IGL NSN 993 DDS GTW 994 V3- D6- 4*01 FNM GGAR KSQ V1- J2* IGN DSS 9* 19* YA GIP 51* 01 NY LSA 01 01 VAG 01 A LEY 60 IGH IGH IGHJ GFT 995 ISFN 996 VKD 997 IGL IGL SSN 998 DDS ATW 999 V3- D6- 4*02 FNM GGAR KSQ V1- J2* IGN DSS 9* 19* YA GIP 51* 01 NY LTA 01 01 LAG 01 A LEY H018 60 IGH IGH IGHJ GFN 1000 ISYN 1001 VKD 1002 IGL IGL NSN 1003 DDS GTW 1004 V3- D6- 4*01 FNM GGAR KSQ V1- J2* IGN DSS 9* 19* YA GIP 51* 01 NF LSA 01 01 VAG 01 A LEY 69 IGH IGH IGHJ GGS 1005 IYYS 1006 AIY 1007 IGKV IGKJ QSI 1008 AVS QQS 1009 V4- D2- 3*02 ITT GST MDE 1-39 2*01 GNY YTI 30- 2* GDY AWA *01 SLF 4* 03 Y FEI T 01 H019 69 IGH IGH IGHJ GGS 1010 IYYS 1011 AIY 1012 IGKV IGKJ QSV 1013 AVS QQS 1014 V4- D2- 3*02 ITT GST MDE 1-39 2*01 GNY YTI 30- 2* GDY AWA *01 SLF 4* 03 Y FEI T 01 146 IGH IGH IGHJ GGS 1015 IYYS 1016 VRE 1017 IGL IGL SSD 1018 EVT SSY 1019 V4- D3- 4*02 ISG GNT NYI V2- J3* VGG AGS 30- 10* GDY TSP 8* 02 YNY NDV 4* 01 Y LSR 01 V 01 146 IGH IGH IGHJ GGS 1020 VYSS 1021 ASY 1022 IGL IGL ALP 1023 EDH YST 1024 V4- D4- 4*02 INS GST TVT V3- J3* KKY DSS 30- 17* GDY TWG 10* 02 GNY 4* 01 Y GFD 01 RV 01 Y 99 IGH IGH IGHJ GGS 1025 IHYS 1026 ARG 1027 IGKV IGKJ QSV 1028 GVS QQY 1029 V4- D4/ 4*02 ISS GST VLH 3-20 2*01 SSS GSS 31* OR GNY *01 Y PYT 02 15- Y 4a* 01 13 IGH IGH IGHJ GGS 1030 IYYS 1031 ARV 1032 IGKV IGKJ QGI 1033 SAS QKY 1034 V4- D3- 4*02 ISS DTTY VSS 1-27 1*01 SNY WT 31* 22* GGY YSGS GHR *01 03 01 Y T HYY FDY 60 IGH IGH IGHJ RGS 1035 ILNT 1036 AQS 1037 IGKV IGKJ SIN 1038 DAS QQY 1039 IGL IGL SGS 1040 EDN QSY 1041 V4- D1- 5*02 VGW GID RRL 3-15 2*03 IN DKW V6- J3* IAS DSS 31* 1* GEN VGP *01 PRS 57* 02 NY NHG 03 01 F FVS 01 V 99 IGH IGH IGHJ GGS 1042 ISYS 1043 ARG 1044 IGKV IGKJ QSV 1045 GAS QQY 1046 V4- D2- 4*02 ISS GST VLV 3-20 2*01 SRA DSS 31* 8* GGY *01 Y PYT 03 02 Y 146 IGH IGH IGHJ GGS 1047 IYYD 1048 ARV 1049 IGKV IGKJ QGI 1050 AAS LQD 1051 IGL IGL SSN 1052 DNY GTW 1053 V4- D3- 3*01 INS GSA VHA 1-6* 4*01 RND YNY V1- J3* IGN DSS 31* 10* DDY SAN 01 PLT 51* 02 TF LNG 03 01 Y AFD 01 WV V 146 IGH IGH IGHJ GGS 1054 IYYD 1055 ARV 1056 IGKV IGKJ QSI 1057 DAS QQT 1058 V4- D3- 3*01 ISN GSA VHA 1-39 5*01 NKF YST 31* 10* DNY SAN *01 PT 03 01 Y AFD V 146 IGH IGH IGHJ GVP 1059 IHAS 1060 ARV 1061 IGKV IGKJ QSV 1062 GAS QQY 1063 V4- D4- 3*02 INN GAT PLR 3-15 4*01 SSD KNW 31* 11* AGF DFY *01 PPL 03 01 Y SNY T SPS AFD I 9 IGH IGH IGHJ RGS 1064 INHS 1065 AGG 1066 IGKV IGKJ QSL 1067 LGS MQA 1068 V4- D3- 5*02 FSD GST RFT 2-28 4*01 LHS LQT 34* 16* YY NDF *01 NGY LLL 01 02 VWG NY T SYR YES 60 IGH IGH IGHJ GGS 1069 ISHS 1070 VRG 1071 IGL IGL SSN 1072 SNN AAW 1073 V4- D6- 4*02 FIG GSA GYS V1- J3* IGS DDS 34* 19* HY SAP 44* 02 NT LNG 01 01 YPR 01 WV EWR Y 69 IGH IGH IGHJ GGS 1074 INQS 1075 ARG 1076 IGKV IGKJ QSV 1077 DGS QQR 1078 V4- D5- 6*03 FSG GST RDG 3-11 1*01 TNY SNW 34* 24* DF YNY *01 QWT 01 01 VGY YYY YYM DV 146 IGH IGH IGHJ GGT 1079 IDHS 1080 ARG 1081 IGKV IGKJ QTI 1082 GAS QQY 1083 IGL IGL SLR 1084 GRN SSR 1085 V4- D3- 6*02 FSG GGT IFE 3-15 3*01 SNN NNW V3- J2* SYY SGN 34* 3* YY VVI *01 PPF 19* 01 RLV 01 01 IPY T 01 YSY RVD V 9 IGH IGH IGHJ GGP 1086 INHS 1087 GRG 1088 IGL IGL SRQ 1089 EVN RSY 1090 V4- D6- 5*02 FSG GST LGR V2- J3* DGR ISN 34* 13* YY EYS 14* 02 YX NXX 02 01 SSW 01 WV YGG RRF DP 9 IGH IGH IGHJ GYS 1091 MYHS 1092 ARD 1093 IGKV IGKJ QSV 1094 GAS QQY 1095 V4- D3- 3*02 IRN GST RSG 3-20 4*01 SSS GSS 38- 22* RYY YVF *01 Y PLT 2* 01 FYD 02 AFD I 146 IGH IGH IGHJ GYS 1096 FSHS 1097 GGG 1098 IGL IGL SSN 1099 TND AVW 1100 V4- D2- 4*02 ISR GTT VTR V1- J3* IGK DDN 38- 21* DYY ADY 47* 02 NY LSA 2* 02 02 WE 02 60 IGH IGH IGHJ GGS 1101 IDYY 1102 ARR 1103 IGKV IGKJ QSI 1104 KAS HQY 1105 V4- D2- 4*02 ISS GST IQL 1-5* 1*01 SSW NTY 39* 8* SSY MVF 03 PWT 01 01 Y DF 60 IGH IGH IGHJ GGS 1106 IYYS 1107 ARH 1108 IGL IGL SST 1109 LNN ASW 1110 V4- D3- 2*01 ISN GST PYY V1- J2* IGS DDS 39* 3* SNY NFW 44* 01 NT LNG 01 01 Y IYW 01 LVV YFD L 99 IGH IGH IGHJ GGS 1111 VSSK 1112 TRH 1113 IGL IGL SSN 1114 ANN AVW 1115 V4- D3- 3*01 IRS GKT WLG V1- J3* IGV DDS 39* 10* SGY GDK 44* 02 NT LNT 01 01 F WSQ 01 WV SPF LAV 99 IGH IGH IGHJ GGS 1116 IYYG 1117 AKG 1118 IGL IGL SNN AAW 1119 V4- D5- 3*02 IST GST RYS V1- J3* DDS 39* 12* SNY GYN 47* 02 PEW 01 01 Y DYN 02 LG AFD I 146 IGH IGH IGHJ GGS 1120 IYYS 1121 TRP 1122 IGKV IGKJ QSV 1123 GAS QQY 1124 V4- D3- 3*01 ISS GTT ASG 3-20 5*01 SSS GSS 39* 10* MSY AHD *01 Y SIT 01 01 Y YVS RSY YPG QGA FGV 146 IGH IGH IGHJ GGS 1125 LYYT 1126 ARL 1127 IG IGL SNN 1128 RNN STW 1129 V4- D6- 4*02 IIS GIT LGI LV J3* IDN DSS 39* 13* YTY AAT 10- 02 QG LST 01 01 Y GHF 54* WL DS 01 146 IGH IGH IGHJ GGS 1130 IYYS 1131 TRP 1132 IGKV IGKJ QSV 1133 GAS QQY 1134 V4- D3- 3*02 ISS GTP ASG 3-20 5*01 STT GSS 39* 10* ISY AHD *01 Y STT 01 01 Y YAS RSY YPG LGA FGI 146 IGH- IGH IGHJ GGS 1135 IYYS 1136 ARP 1137 IGKV IGKJ QSV 1138 GAS QQH 1139 V4- D3- 3*02 ITS GTT LLN 3-20 4*01 SSK DNS 39* 3* LSY PMT *01 C LS 01 01 W LYG VTP GIG PFE I 146 IGH IGH IGHJ GDS 1140 INYN 1141 AAH 1142 IGKV IGKJ QNI 1143 AAS QQS 1144 V4- D6- 4*02 MSR GIT RVS 1-39 1*01 DDY YNT 39* 6* NSF SSY *01 PT 01 01 Y PAD Y 146 IGH IGH IGHJ GGS 1145 IYYS 1146 ARP 1147 IGKV IGKJ QSI 1148 GAS QQY 1149 V4- D3- 3*02 SIS GTA LLN 3D- 1*01 RSN INW 39* 3* LSY PST 15* PPW 01 01 Y IYG 01 T VTP GIG PFE M 146 IGH IGH IGHJ GYS 1150 IYHI 1151 ARG 1152 IGKV IGKJ QSI 1153 LAS QRS 1154 V4- D3- 6*02 VST GST NYD 1-39 2*01 DNY YST 4* 16* SNW YVW *01 PYT 02 02 GSY RSD QGY GLD V 49 IGH IGH IGHJ GAS 1155 VHSS 1156 ARE 1157 IGL IGL SSD 1158 DVT SSY 1159 V4- D6- 6*03 IRS GGT GGS V2- J2* VGS AGI 4* 13* HY SYY 8* 01 YNY NSY 07 01 YYY 01 VI YMD V 13 IGH IGH IGHJ GGS 1160 IYYN 1161 ARS 1162 IGKV IGKJ QSV 1163 GAS QQY 1164 V4- D2- 5*02 IST GGT KNQ 3-15 4*01 GSD NDW 59* 2* YF LLL *01 PPL 01 01 FDP T 13 IGH IGH IGHJ GDS 1165 VYHT 1166 ARS 1167 IGKV IGKJ QDI 1168 DAS QQY 1169 V4- D4- 5*01 IGT GGT KNQ 1-33 4*01 SNY DNL 59* 23* YF LLL *01 PLT 01 01 FEF 55 IGH IGH IGHJ GAS 1170 MYSS 1171 ART 1172 IGKV IGKJ QNI 1173 KAS QQY 1174 IGL IGL NNN 1175 ED SAW 1176 V4- D7- 3*01 ISS GSV NWA 1-5* 1*01 NSW YSY V1- J2* IGR DFS 59* 27* NY YDP 03 ST 36* 01 SA LSV 01 01 FNV 01 QV 60 IGH IGH IGHJ GGS 1177 IYDS 1178 ARD 1179 IGKV IGKJ QSI 1180 KAS QQY 1181 V4- D2- 6*02 ISS GST RGY 1-5* 4*01 SRW NSY 59* 15* YY CSG 03 FPL 01 01 GSC T LGG MDV 60 IGH IGH IGHJ GGS 1182 IYDS 1183 VRD 1184 IGKV IGKJ QSI 1185 KAS QQY 1186 V4- D2- 6*02 ISG GNT RGF 1-5* 1*01 SSW NSY 59* 8* SY CTG 03 RT 01 02 KSC LGG MDV 60 IGH IGH IGHJ GGS 1187 VYSS 1188 ARL 1189 IGL IGL TSN 1190 INN AAW 1191 V4- D2- 4*02 ISN GTT RRR V1- J2* IGD DDS 59* 8* YF GLT 44* 01 NN LNG 01 02 GTD 01 PNV FDY V 69 IGH IGH IGHJ GGS 1192 IYYS 1193 ARS 1194 IGL IGL KLG 1195 QDT QAW 1196 V4- D3- 6*03 IRS GST YYY V3- J2* DKY DSS 59* 22* YY DSS 1* 01 VV 01 01 GYR 01 PSF YYY YMD V 146 IGH IGH IGHJ GGS 1197 IYYS 1198 ARG 1199 IGKV IGKJ QSI 1200 ATS HQS 1201 V4- D1- 6*02 ISG GTT ILG 1-39 2*01 SSY YSS 59* 26* YY STW *01 PYT 01 01 YYY YGL DV 55 IGH IGH IGHJ GGS 1202 IHSK 1203 ARH 1204 IGKV IGKJ QGI 1205 AAS QQA 1206 V4- D5- 4*02 ISN GDT LYR 1-12 4*01 SSG NSF 59* 18* DY YGY *01 PLT 08 01 RNY FDY H020 55 IGH IGH IGHJ GGS 1207 IHSK 1208 ARH 1209 IGKV IGKJ QGI 1210 AAS QQA 1211 V4- D5- 4*02 ISN GDT LYR 1-12 4*01 SSG NSF 59* 18* DY YGY *01 PLT 08 01 RNY FDY 69 IGH IGH IGHJ GGS 1212 IYTG 1213 ARM 1214 IGKV IGKJ QTI 1215 EAS QEY 1216 V4- D6- 3*02 VRS GAT TSF 1-5* 2*01 GTY NSY 61* 13* TGY KQS 01 SYT 08 01 F GGW YRG RHD GFD I 69 IGH IGH IGHJ RGS 1217 VYYT 1218 ARL 1219 IGKV IGKJ QSI 1220 DGS QEY 1221 V4- D6- 3*02 VSN GSS TSY 1-5* 2*01 STL SSY 61* 19* GGY KQR 01 SYT 08 01 Y GGW YRG RHD AFD I 9 IGH IGH IGHJ GYS 1222 IQSG 1223 ARR 1224 IGL IGL SSD 1225 DVT SSY 1226 V5- D3- 4*02 FTS DYNT ARN V2- J3* VGR ISS 51* 22* YW VGN 14* 02 YNY NTL 01 01 YGT 01 WV SDF YPY FDH 60 IGH IGH IGHJ GYT 1227 INPP 1228 ARR 1229 IGL IGL SSD 1230 EVN SSY 1231 V5- D1- 3*02 FAS NSDT RVS V2- J2* VGG AGT 51* 14* YW VTG 8* 01 YNY NTF 01 01 TDA 01 VV FDI 60 IGH IGH IGHJ GYT 1232 INPP 1233 ARR 1234 IGL IGL SSD 1235 EVN SSY 1236 V5- D1- 3*02 FAS NSDT RVS V2- J2* VGG AGT 51* 14* YW VTG 8* 01 YNY NTF 01 01 TDA 01 VV FDI 69 IGH IGH IGHJ GDT 1237 ILLS 1238 ARA 1239 IGL IGL SSD 1240 DVT SSY 1241 V5- D1- 4*02 FGN DSDT TPG V2- J1* VGA TDS 51* 1* YW NYY 14* 01 YNY SPN 01 01 FDS 01 CV 99 IGH IGH IGHJ GYS 1242 IYPG 1243 ARP 1244 IGKV IGKJ QSV 1245 GAS QQY 1246 V5- D2- 1*01 FSN DSDT SRS 3-20 4*01 SSR ANS 51* 2* FW RDI *01 S PLT 01 01 NKW YLS TSE YFH Y 9 IGH IGH IGHJ GDS 1247 TYYR 1248 AVG 1249 IGL IGL SSD 1250 EVS SSH 1251 V6- D1- 4*02 VSN SKWF HHW V2- J1* VGG AGS 1* 1* NTA N HFK 8* 01 YSH NYG 01 01 V Y 01 V 9 IGH IGH IGHJ GDS 1252 TYYR 1253 AVG 1254 IGL IGL SSD 1255 EVS SSH 1256 V6- D1- 4*02 VSN SKWF HHW V2- J1* VGG AGS 1* 1* NTA N HFK 8* 01 YSH NYG 01 01 V Y 01 V 55 IGH IGH IGHJ GSS 1257 TYYR 1258 ARD 1259 IGKV IGKJ ESI 1260 AAS QQS 1261 V6- D3- 4*02 IFT SKWY TYY 1-39 5*01 RSN YRT 1* 10* NSA N YTS *01 PIT 01 01 G ASY YNV DY 55 IGH IGH IGHJ GYT 1262 INTD 1263 ARL 1264 IGL IGL SSN 1265 GNN QSY 1266 V7- D1- 4*02 FTS TGNP GEY V1- J2* IGA DRS 4- 7* YG SWN 40* 01 GYD LIL 1* 01 SIG 01 VV 02 YFD Y 99 IGH IGH IGHJ GYV 1267 INTN 1268 ARS 1269 IGKV IGKJ QNI 1270 EAS QQS 1271 V7- D3- 4*02 FTN TGNP YAY 1-39 1*01 AIR DTL 4- 10* YA GDF *01 PWT 1* 01 02 99 IGH IGH IGHJ GYT 1272 INTN 1273 ARG 1274 IGKV IGKJ QSV 1275 WAS QQY 1276 V7- D3- 4*02 FSN TGNP ARS 4-1* 1*01 LYR YNT 4- 22* YA YYD 01 SNN LTW 1* 01 SSG KNY A 02 YYS WSD Y

TABLE S3 Primers and synthesized nucleotide sequences. Single-Cell Antibody Cloning Primers A2 SEQ ID NO: F1-HC 5′-ACAGGTGCCCACTCCCAGGTGCAG 1277 F2-HC 5′-AAGGTGTCCAGTGTGARGTGCAG 1278 F3-HC 5′-CCCAGATGGGTCCTGTCCCAGGTGCAG 1279 F4-HC 5′-CAAGGAGTCTGTTCCGAGGTGCAG 1280 R-1st-HC 5′-GGAAGGTGTGCACGCCGCTGGTC 1281 R-2nd-HC 5′-GTTCGGGGAAGTAGTCCTTGAC 1282 F1-Kappa 5′-ATGAGGSTCCCYGCTCAGCTGCTGG 1283 F2-Kappa 5′-CTCTTCCTCCTGCTACTCTGGCTCCCAG 1284 F3-Kappa 5′-ATTTCTCTGTTGCTCTGGATCTCTG 1285 F4-Kappa 5′-ATGACCCAGWCTCCABYCWCCCTG 1286 R-1st-Kappa 5′-GTTTCTCGTAGTCTGCTTTGCTCA 1287 R-2nd- 5′-GTGCTGTCCTTGCTGTCCTGCT 1288 Kappa F1-Lambda 5′-GGTCCTGGGCCCAGTCTGTGCTG 1289 F2-Lambda 5′-GGTCCTGGGCCCAGTCTGCCCTG 1290 F3-Lambda 5′-GCTCTGTGACCTCCTATGAGCTG 1291 F4-Lambda 5′-GGTCTCTCTCSCAGCYTGTGCTG 1292 F5-Lambda 5′-GTTCTTGGGCCAATTTTATGCTG 1293 F6-Lambda 5′-GGTCCAATTCYCAGGCTGTGGTG 1294 F7-Lambda 5′-GAGTGGATTCTCAGACTGTGGTG 1295 R-1st- T-CACCAGTGTGGCCTTGTTGGCTTG 1296 Lambda R-2nd- 5′-CTCCTCACTCGAGGGYGGGAACAGAGTG 1297 Lambda F-Vector 5′-GCTTCGTTAGAACGCGGCTAC 1298 Seq Primer Antibody Cloning Primers for Each Antibody F-H001-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1299 CAGGTCCAGCTTGTGCAGTCTGG R-H001-HC 5′-CCGATGGGCCCTTGGTCGACGC 1300 TGAAGAGACGGTGACCATTGTCCCTT F-H001- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT GACATCCAGATGA 1301 Kappa CCCAGTCTCCA R-H001- 5′-GAAGACAGATGGTGCAGCCACCGTACG TTTGATCTCCACCTTG 1302 Kappa GTCCCTCC F-H002-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1303 CAGGTCACCTTGAAGGAGTCTGG R-H002-HC 5′-CCGATGGGCCCTTGGTCGACGC 1304 TGAGGAGACGGTGACCAGGGTG F-H002- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATGTGCTGA 1305 Lambda CCCAGGCGC F-H003-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1306 GAGATGCAGCTGCTGGAGTCTGG R-H003-HC 5′-CCGATGGGCCCTTGGTCGACGC 1307 TGAGGAGACAGTGACCAGGGTGC F-H003- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATATGCTGA 1308 Lambda CTCAGGCACCC F-H004-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1309 GAGATGCAACTGGTGGAGTCTGG R-H004-HC 5′-CCGATGGGCCCTTGGTCGACGC 1310 TGAGGAGACGATGACCGTGGTCC F-H004- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATGTGCTGA 1311 Lambda CTCAGCCACC F-H005-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1312 CAGATGCGTCTGGTGGAATCTGG R-H005-HC 5′-CCGATGGGCCCTTGGTCGACGC 1313 TGAGGAGACGGTGACCGGGGT F-H005- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATGTGCTGA 1314 Lambda CTCAGCCACC F-H006-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1315 CAGATGCGTCTGGTGGAATCGGG R-H006-HC 5′-CCGATGGGCCCTTGGTCGACGC 1316 TGAGGAGACGGTGACCGGGATC F-H006- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATGTGCTGA 1317 Lambda CTCAGCCACC F-H007-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1318 CAGGTGCACCTGGTGGAGTCTG R-H007-HC 5′-CCGATGGGCCCTTGGTCGACGC 1319 TGAGGAGACGGTGACCGTGGTC F-H007- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT GAAATTGTGTTG 1320 Kappa ACGCAGTCTCCAG R-H007- 5′-GAAGACAGATGGTGCAGCCACCGTACG TTTGATCTCCAACTTG 1321 Kappa GTCCCCTGG F-H008-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1322 CAGATGCACCTATTGGAGTCTGGG R-H008-HC 5′-CCGATGGGCCCTTGGTCGACGC 1323 TGACGAGACGGTGACCCTGGTC F-H008- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATGTGCTGA 1324 Lambda CTCAGCCACC F-H009-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1325 CAGATGAAGTTGGTGGAGTCTGGG R-H009-HC 5′-CCGATGGGCCCTTGGTCGACGC 1326 TGAGGAGACGGTGACCGTGGTC F-H009- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATGTGCTGA 1327 Lambda CTCAGCCACC F-H010-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1328 CAGGTGCAGCTGGTGGAGTCTG R-H010-HC 5′-CCGATGGGCCCTTGGTCGACGC 1329 TGAGGAGACGGTGACCAGGGTT F-H010- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATGTGCTGA 1330 Lambda CTCAGCCACC F-H011-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1331 CAGGTTCACTTGGCGGAGTCTGG R-H011-HC 5′-CCGATGGGCCCTTGGTCGACGC 1332 TGAAGAGACGGTGACCAATGTCCC F-H011- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT GAAGTTGTGTTGA 1333 Kappa CACAGTCTCCAGC R-H011- 5′-GAAGACAGATGGTGCAGCCACCGTACG TTTGATCTCCAGCTTG 1334 Kappa GTCCCCTG F-H012-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1335 CAGCTGCAGCTGGTGGAGTCTG R-H012-HC 5′-CCGATGGGCCCTTGGTCGACGC 1336 TGAGGAGACGGTGACCAGGGTTC F-H012- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATGTGCTGA 1337 Lambda CTCAGCCACC F-H013-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1338 CAGGTGCAGCTGGTGGAGTCTG R-H013-HC 5′-CCGATGGGCCCTTGGTCGACGC 1339 TGAGGAGACGGTGACCAGGGCT F-H013- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATGTGCTGA 1340 Lambda CTCAGCCACC F-H014-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1341 CAGGTACAACTGATGGAGTCTGGG R-H014-HC 5′-CCGATGGGCCCTTGGTCGACGC 1342 TGAGGAGACGGTGACCAGGGCT F-H014- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC TCCTATGTGCTGA 1343 Lambda CTCAGACACCC F-H015-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1344 CAGGTGCAGCTGGTGGAGTCTG R-H015-HC 5′-CCGATGGGCCCTTGGTCGACGC 1345 TGAGGAGACGGTGACCAGGGTTC F-H015- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT GACATCCAGGTGA 1346 Kappa CCCAGTCAC R-H015- 5′-GAAGACAGATGGTGCAGCCACCGTACG TTTGATGTCCACCTTG 1347 Kappa GTCCCTCC F-H016-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1348 GAGATGCACCTGGTGGAGTCTGG R-H016-HC 5′-CCGATGGGCCCTTGGTCGACGC 1349 TGAGGAGACAGTGACCAGGGTGC F-H016- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT GAAATACTGCTGA 1350 Kappa CGCAGTCTCCAG R-H016- 5′-GAAGACAGATGGTGCAGCCACCGTACG TTTGATCTCCACCTTG 1351 Kappa GTCCCTCC F-H017-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1352 GAGGTGCAGCTGGTGGAGTCC R-H017-HC 5′-CCGATGGGCCCTTGGTCGACGC 1353 TGAGGAGACGGTGACCAGGGTTC F-H017- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC CAGTCTGCCCTGA 1354 Lambda CTCAGCCTG F-H018-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1355 GAAGGACAGCTGGTGGAGTCTGG R-H018-HC 5′-CCGATGGGCCCTTGGTCGACGC 1356 TGAGGAGACGGTGACCAGGGTTC F-H018- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCC CAGTCTGTGTTGA 1357 Lambda CGCAGCCGC F-H019-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1358 CAGGTGGTGCTGCAGGAGTCG R-H019-HC 5′-CCGATGGGCCCTTGGTCGACGC 1359 TGAAGAGACGGCGACCAGTGTCC F-H019- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT GACATCCAGATGA 1360 Kappa CCCAGTCTCCG R-H19- 5′-GAAGACAGATGGTGCAGCCACCGTACG TTTGATCTCCAGCTTG 1361 Kappa GTCCCCTG F-H020-HC 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT 1362 CAGGTGCAGCTGCAGGAGTCG R-H020-HC 5′-CCGATGGGCCCTTGGTCGACGC 1363 TGAGGAGACGGTGACCAGGTTTCC F-H020- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCT GACATCCAGATGA 1364 Kappa CCCAGTCTCCA R-H020- 5′-GAAGACAGATGGTGCAGCCACCGTACG TTTGAACTCCACCTTG 1365 Kappa GTCCCTCC R-Lambda 5′-GGCTTGAAGCTCCTCACTCGAGGGYGGGAACAGAGTG 1366 gBlock Synthesis for Alanine Mutations 101Q 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1367 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATGCAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 102G 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1368 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGCAATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 103M 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1369 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTGCATTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 104L 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1370 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGGCACCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 105P 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1371 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGGCAGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 106V 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1372 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGCATGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 108P 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1373 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTGCACT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 109L 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1374 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTGC AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 110I 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1375 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AGCACCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 111P 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1376 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTGCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 112G 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1377 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGCATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 113S 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1378 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGAGCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 114T 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1379 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAGCAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 115T 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1380 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAGCAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 116T 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1381 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAGCAAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 117S 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1382 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCGCAACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 118T 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1383 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTGCAGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 119G 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1384 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGCACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 120P 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1385 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGAGCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 122K 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1386 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCGCAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 123T 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1387 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAGCA TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 125T 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1388 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCGCAACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 126T 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1389 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGGCACCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 127P 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1390 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTGCAGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 129Q 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1391 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTGCAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 130G 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1392 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGCAAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 131N 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1393 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCGCATCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 132S 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1394 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACGCAATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 133M 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1395 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTGCATTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 134F 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1396 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGGCACCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 135P 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1397 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTGCATCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 136S 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1398 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCGCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 140T 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1399 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTGCAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 141K 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1400 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAGCACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 142P 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1401 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAAGCAACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 143T 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1402 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTGCAGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 144D 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1403 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGCAGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 145G 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1404 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGCAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 146N 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1405 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAGCATGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 148T 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1406 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCGCATGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 150I 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1407 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTGCACCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 151P 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1408 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTGCAAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 152I 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1409 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCGC ACCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 153P 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1410 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CGCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 154S 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1411 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCAGCATCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 155S 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1412 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGGCATGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 156W 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1413 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCGCAGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 158F 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1414 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTGCAGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 160K 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1415 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAGCATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 161Y 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1416 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAAGCACTATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 162L 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1417 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACGCATGGGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 163W 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1418 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTAGCAGAGTGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 164E 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1419 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGCATGGGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 165W 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1420 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGGCAGCC TCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 167S 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1421 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC GCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 168V 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1422 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGCACGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 169R 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1423 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCGCATTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 170F 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1424 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTGCATCTTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 171S 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1425 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCGCATGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG 172W 5′-ACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGA 1426 CTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCA AAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTC CTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTT TATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTAT TGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCT AATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTT GCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT CCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTCCGTTTCTCTGCACTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGG gBlock Synthesis for Germline Antibodies H006- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGG 1427 HC_GL TGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGC ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAG ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAA GAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGAC ACGGCTGTGTATTACTGTGCGAAAGATGCTTATCTTTCTGCAG CGAGAGGATACGGTATGGACGTCTGGGGCCAAGGGACCACGGT CACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGG H006- 5′-CTAGTAGCAACTGCAACCGGTTCCTGGGCCTCCTATGTGCTGA 1428 Lambda_GL CTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAG GATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCAC TGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCT ATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTC TGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGG GTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGG ATAGTAGTAGTGATCATGTGGTATTCGGCGGAGGGACCAAGCT GACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGT TCCCACCCTCGAGTGAGGAGCTTCAAGCC H019- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGC 1429 HC_GL AGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTC CCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGAT TACTACTGGAGTTGGATCCGCCAGCCCCCAGGGAAGGGCCTGG AGTGGATTGGGTACATCTATTACAGTGGGAGCACCTACTACAA CCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCC AAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCAG ACACGGCCGTGTATTACTGTGCCATCTACATGGATGAGGCCTG GGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCT TCAGCGTCGACCAAGGGCCCATCGG H019- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCTGACATCCAGATGA 1430 Kappa_GL CCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTC ACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAA ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGAT CTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA GTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAG TTACAGTATTTCCTTATTCACTTTTGGCCAGGGGACCAAGCTG GAGATCAAACGTACGGTGGCTGCACCATCTGTCTTC H020- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCTCAGGTGCAGCTGC 1431 HC_GL AGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTC CCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACT GGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGAT TGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCC CTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACC AGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGC CGTGTATTACTGTGCGAGACACCTTTATCGCTATGGTTATAGG AACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCAGCGTCGACCAAGGGCCCATCGG H020- 5′-CTAGTAGCAACTGCAACCGGTGTACATTCTGACATCCAGATGA 1432 Kappa_GL CCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGT CACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTA GCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGA TCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTT CAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC AGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGG CTAACAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGA GATCAAACGTACGGTGGCTGCACCATCTGTCTTC HBV TaqMan PCR F-sense 5′-CCGTCTGTGCCTTCTCATCTG 1433 R-anti- 5′-AGTCCAAGAGTCCTCTTATGTAAGACCTT 1434 sense Probe 5-/56 FAM/CCGTGTGCA/ZEN/CTTCGCTTC ACCTCT 1435 GC/3IABkFQ/-3 S-protein PCR F-S-protein 5′-CCCTGCGCTGAACATGGAGAACA 1436 R-S-protein 5′-AAATGTATACCCAAAGACAAAAGAAAA 1437

It will be recognized from the foregoing that the present disclosure describes screening individuals who were either vaccinated or had spontaneously recovered from HBV infection. Antibody cloning from memory B cells revealed that all 5 of the top individuals produced clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Clones with the same immunoglobulin variable, diversity and joining heavy and light chain genes were shared among elite neutralizers. Single bNAbs protected humanized mice against infection, but selected for resistance mutations in mice with established infection. In contrast, infection was controlled in the absence of detectable escape mutations by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with a peptide epitope containing residues frequently mutated in human immune escape variants revealed a loop anchored by oppositely charged residues. The structure provides a molecular explanation for why immunotherapy for chronic HBV infection may require combinations of complementary bNAbs, as described herein.

While the disclosure has been described through specific embodiments, routine modifications will be apparent to those skilled in the art and such modifications are intended to be within the scope of the present disclosure. 

1. An isolated or recombinant antibody or antigen binding fragment thereof, said isolated or recombinant antibody or antigen binding fragment thereof comprising complementarity determining regions (CDRs), the CDRs comprising heavy and light chain amino acid sequences CDR1, CDR2 and CDR3 selected from the antibody heavy and light chain CDRs of Table S2.
 2. The recombinant or isolated antibody or antigen binding fragment thereof of claim 1, comprising the heavy and light chain CDR1, CDR2 and CDR3 sequences of antibody H017 from Table S2, or the heavy and light chain CDR1, CDR2 and CDR3 sequences of antibody H019 from Table S2, the heavy and light chain CDR1, CDR2 and CDR3 sequences of antibody H016 from Table S2.
 3. The recombinant or isolated antibody or antigen binding fragment thereof of claim 1, comprising the heavy and light chain CDR1, CDR2 and CDR3 sequences of H004 from Table S2, or the heavy and light chain CDR1, CDR2 and CDR3 sequences of H005 from Table S2, or the heavy and light chain CDR1, CDR2 and CDR3 sequences of H008 from Table S2, or the heavy and light chain CDR1, CDR2 and CDR3 sequences of H009 from Table S2.
 4. The recombinant or isolated antibody of claim 1, comprising at least one modification of its constant region, wherein the modification increases in vivo half-life of the antibody, or alters the ability of the antibody to bind to Fc receptors, or inhibits aggregation of the antibodies, or a combination of said modifications, or wherein the antibody is attached to a detectable label or a substrate.
 5. The recombinant or isolated antibody of claim 4, comprising the modification that increases in vivo half-life of the antibody.
 6. The recombinant or isolated antibody of claim 4, comprising the modification that alters the ability of the antibody to bind to Fc receptors.
 7. A pharmaceutical composition comprising an antibody or an antigen binding fragment thereof, or a combination of antibodies or antigen binding fragment thereof, of claim
 1. 8. The pharmaceutical composition of claim 7, comprising the combination of the antibodies.
 9. The pharmaceutical composition of claim 8, wherein the combination of the antibodies includes the H017 and H019 antibodies.
 10. The composition of claim 9, wherein the composition further comprises the H016 antibody.
 11. A method for prophylaxis or therapy of a Hepatitis B virus infection comprising administering to an individual in need thereof an effective amount of at least one antibody or antigen binding fragment thereof of claim 1, wherein optionally the at least one antibody comprises at least one modification of the constant region.
 12. The method of claim 11, wherein the administering comprises administering a combination of antibodies that include the H017 and H019 antibodies.
 13. The method of claim 12, wherein the administration further comprises administering the H016 antibody.
 14. The method of claim 11, comprising administering a combination of at least two of the antibodies, and wherein administering the combination of at the least two antibodies provides a therapeutic and/or prophylactic effect against infection by a hepatitis B virus that comprises one or more escape mutations in at least one of the HBsAg or the S-protein of the hepatitis B virus.
 15. The method of claim 14, wherein the combination of at least two of the antibodies comprises the H017 and H019 antibodies.
 16. The method of claim 15, wherein the combination of at least two of the antibodies further comprises the H016 antibody.
 17. One or more recombinant expression vectors encoding the heavy chain and the light chain of any one of the antibodies or antigen binding fragments thereof of claim
 1. 18. Cells comprising one or more recombinant expression vectors of claim
 17. 19. A method comprising culturing cells of claim 18 and separating antibodies from the cells.
 20. A kit comprising one or more expression vectors encoding according to claim
 17. 21. A method for detecting Hepatitis B virus comprising: contacting a biological sample from an individual with an antibody of claim 1, and detecting the presence of a complex comprising the antibody and a Hepatitis B virus protein.
 22. A method comprising testing one or more candidate drug agents for the capability to target an antigenic loop region of the hepatitis B virus S-protein by interfering with a complex formed by the peptide and an antibody of claim
 1. 23. A vaccine comprising at least two non-overlapping epitopes from the Hepatitis B virus S antigen (HBsAg), wherein optionally at least one of the non-overlapping epitopes does not comprise a commonly occurring escape mutation.
 24. The vaccine of claim 23, comprising at least three, non-overlapping epitopes from the HBsAg.
 25. A method for prophylaxis and/or therapy for Hepatitis B virus infection comprising administering a vaccine of claim 23 to an individual in need thereof.
 26. The method of claim 25, wherein the administering comprises at least three, non-overlapping epitopes from the HBsAg.
 27. The method of claim 26, wherein the Hepatitis B virus in the individual does not develop Hepatitis B virus comprising escape mutations subsequent to administering the vaccine. 